Ketoreductases and uses thereof

ABSTRACT

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize a variety of chiral compounds.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §119(e) of applicationSer. No. 60/900,494, filed Feb. 8, 2007 and application Ser. No.60/900,430, filed Feb. 8, 2007, the contents of which are incorporatedherein by reference.

2. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing submitted concurrently herewith under 37 C.F.R.§§1.821 in a computer readable form (CRF) as file name 376247-015US.txtis herein incorporated by reference in its entirety. The electronic copyof the Sequence Listing was created on Feb. 7, 2008 with a file size of221 KB.

3. BACKGROUND

Enzymes belonging to the ketoreductase (KRED) or carbonyl reductaseclass (EC1.1.1.184) are useful for the synthesis of optically activealcohols from the corresponding prochiral ketone substrate. KREDstypically convert a ketone substrate to the corresponding alcoholproduct, but may also catalyze the reverse reaction, oxidation of analcohol substrate to the corresponding ketone/aldehyde product. Thereduction of ketones and the oxidation of alcohols by enzymes such asKRED requires a co-factor, most commonly reduced nicotinamide adeninedinucleotide (NADH) or reduced nicotinamide adenine dinucleotidephosphate (NADPH), and nicotinamide adenine dinucleotide (NAD) ornicotinamide adenine dinucleotide phosphate (NADP) for the oxidationreaction. NADH and NADPH serve as electron donors, while NAD and NADPserve as electron acceptors. It is frequently observed thatketoreductases and alcohol dehydrogenases accept either thephosphorylated or the non-phosphorylated co-factor (in its oxidized andreduced state), but not both.

KRED enzymes can be found in a wide range of bacteria and yeasts (forreviews: Kraus and Waldman, Enzyme catalysis in organic synthesis, Vols.1 & 2, VCH Weinheim 1995; Faber, K., Biotransformations in organicchemistry, 4th Ed. Springer, Berlin Heidelberg N.Y. 2000; Hummel andKula Eur. J. Biochem. 1989 184:1-13). Several KRED gene and enzymesequences have been reported, e.g., Candida magnoliae (Genbank Acc. No.JC7338; GI:11360538) Candida parapsilosis (Genbank Acc. No. BAA24528.1;GI:2815409), Sporobolomyces salmonicolor (Genbank Acc. No. AF160799;GI:6539734).

In order to circumvent many chemical synthetic procedures for theproduction of key compounds, ketoreductases are being increasinglyemployed for the enzymatic conversion of different keto substrates tochiral alcohol products. These applications can employ whole cellsexpressing the ketoreductase for biocatalytic ketone reductions, orpurified enzymes in those instances where presence of multipleketoreductases affects specificity and yield of the desired product. Forin vitro applications, a co-factor (NADH or NADPH) regenerating enzymesuch as glucose dehydrogenase (GDH), formate dehydrogenase etc. is usedin conjunction with the ketoreductase. Examples using ketoreductases togenerate useful chemical compounds include asymmetric reduction of4-chloroacetoacetate esters (Zhou, J. Am. Chem. Soc. 1983 105:5925-5926;Santaniello, J. Chem. Res. (S) 1984:132-133; U.S. Pat. No. 5,559,030;U.S. Pat. No. 5,700,670 and U.S. Pat. No. 5,891,685); reduction ofdioxocarboxylic acids (e.g., U.S. Pat. No. 6,399,339); reduction oftert-butyl (S) chloro-5-hydroxy-3-oxohexanoate (e.g., U.S. Pat. No.6,645,746 and WO 01/40450); reduction pyrrolotriazine-based compounds(e.g., US application No. 2006/0286646); reduction of substitutedacetophenones (e.g., U.S. Pat. No. 6,800,477); and reduction ofhydroxythiolanes (WO 2005/054491).

It is desirable to identify other ketoreductase enzymes that can be usedto carryout conversion of various keto substrates to its correspondingchiral alcohol products.

4. SUMMARY

The present disclosure provides engineered or recombinant ketoreductasepolypeptides capable of reducing or converting the compound1-[4-(4-fluoro2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-propan-2-oneto the corresponding product (R)1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-propan-2-ol.The engineered or recombinant ketoreductases are also capable ofreducing or converting acetophenone to (R)-1-phenylethanol. In theembodiments herein, the engineered ketoreductases have one or moreimproved properties in converting the substrates to the product ascompared to the naturally-occurring wild-type ketoreductase enzymes ofLactobacillus kefir and Lactobacillus brevis.

In one aspect, the recombinant polypeptide capable of converting the1-[4-(4-fluoro2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-propan-2-oneto the corresponding product(R)-1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-propan-2-olcomprises an amino acid sequence having (1) an aromatic amino acid or Gat the amino acid residue corresponding to residue 94 of SEQ ID NO:2 orSEQ ID NO:4, and/or (2) an amino acid residue other than S and N at theamino acid residue corresponding to residue 96 of SEQ ID NO:2 or SEQ IDNO:4.

In some embodiments, the ketoreductase polypeptide comprises an aminoacid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2 orSEQ ID NO:4 with the proviso that the residues corresponding to residue94 is an aromatic amino acid residue or G. The engineered ketoreductasemay optionally include one or more conservative substitutions at otherresidue positions within the amino acid sequence.

In some embodiments, the ketoreductase polypeptides comprise an aminoacid sequence having at least 90% amino acid sequence identity to SEQ IDNO:2 or SEQ ID NO:4 and comprise an aromatic amino acid or G at theamino acid residue corresponding to residue 94 of SEQ ID NO:2 or SEQ IDNO:4.

In some embodiments, the ketoreductase polypeptide capable of convertingthe1-[4-(4-fluoro2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-propan-2-oneto the corresponding product(R)-1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-propan-2-olcan comprise a region having an amino acid sequence that is at leastabout 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a region or domain thereof of SEQ ID NO:2 orSEQ ID NO:4, such as residues 90-233, with the proviso that the aminoacid residue corresponding to residue 94 is an aromatic amino acidresidue or G. In some embodiments of these ketoreductase polypeptides,one or more of the remaining residues corresponding to residues 90-233of SEQ ID NO:2 or SEQ ID NO:4 may have a conservative substitution.

The ketoreductase polypeptide capable of converting the compound1-[4-(4-fluoro2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-propan-2-oneto the corresponding product (R)1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-propan-2-olcan comprise, in addition to an aromatic amino acid or G at the aminoacid residue corresponding to residue 94 of SEQ ID NO:2 or SEQ ID NO:4,one or more of the features selected from: residue 96 is any amino acidother than S/N; residue 153 is an aliphatic amino acid residue otherthan L; residue 199 is any amino acid residue other than L; residue 202is G or an aliphatic amino acid residue other than A; and residue 206 isan aromatic amino acid residue.

In some embodiments, the ketoreductase polypeptide with the specifiedamino acid at residue 94 can comprise on or more of the followingadditional features selected from: residue 49 is a polar amino acidresidue other than K; residue 53 is an acidic amino acid residue;residue 54 is a small or aliphatic amino acid residue other than T/P;residue 60 is an aliphatic amino acid residue other than V; residue 95is an aliphatic amino acid other than V; residue 97 is a small aminoacid or G; residue 109 is a basic amino acid residue other than K;residue 147 is an aliphatic amino acid residue; residue 165 is ahydroxyl or small amino acid residue; residue 197 is a small amino acidresidue or G; residue 223 is an aliphatic amino acid residue other thanL; and residue 233 is a small amino acid residue or G.

In another aspect, the recombinant ketoreductase polypeptide capable ofconverting the compound1-[4-(4-fluoro2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-propan-2-oneto the corresponding product (R)1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-propan-2-olcomprise an amino acid sequence having an amino acid other than S and Nat the amino acid residue corresponding to residue 96 of SEQ ID NO:2 orSEQ ID NO:4. In some embodiments, these recombinant ketoreductasepolypeptides can comprise an amino acid sequence with a G, F, Y, or I atthe amino acid residue corresponding to residue 96 of SEQ ID NO:2 or SEQID NO:4.

In some embodiments, the ketoreductase polypeptide comprises an aminoacid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2 orSEQ ID NO:4 with the proviso that the residue corresponding to residue96 is an amino acid residue other than S and N. The engineeredketoreductase may optionally include one or more conservativesubstitutions at other residue positions within the amino acid sequence.

In some embodiments, these ketoreductase polypeptides can comprise anamino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2or SEQ ID NO:4 with the proviso that the residue corresponding toresidue 96 is G, F, Y, or I.

In some embodiments, the ketoreductase polypeptide capable of converting1-[4-(4-fluoro2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-propan-2-oneto the corresponding product(R)-1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-propan-2-olcan comprise a region having an amino acid sequence that is at leastabout 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a region or domain thereof of SEQ ID NO:2 orSEQ ID NO:4, such as residues 90-233, with the proviso that the residuecorresponding to residue 96 is an amino acid residue other than S and N.In some embodiments of these ketoreductase polypeptides, one or more ofthe remaining residues corresponding to residues 90-233 of SEQ ID NO:2or SEQ ID NO:4 may have a conservative substitution.

In some embodiments, the ketoreductase polypeptide comprises an aminoacid sequence having at least 90% amino acid sequence identity to SEQ IDNO:2 or SEQ ID NO:4 and comprise a G, F, Y, or I at the amino acidresidue corresponding to residue 96 of SEQ ID NO:2 or SEQ ID NO:4. Theseengineered ketoreductase may optionally include one or more conservativemutations at other residue positions within the polypeptide sequence.

In some embodiments, the ketoreductase polypeptide capable of convertingthe compound1-[4-(4-fluoro2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-propan-2-oneto the corresponding product (R)1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-propan-2-olcan comprise, in addition to G, F, Y, or I at the amino acid residuecorresponding to residue 96 of SEQ ID NO:2 or SEQ ID NO:4, one or moreof the features selected from: residue 94 is an aromatic amino acidresidue or G; residue 153 is an aliphatic amino acid residue other thanL; residue 199 is any amino acid residue other than L; residue 202 is analiphatic amino acid residue other than A; and residue 206 is anaromatic amino acid residue.

In some embodiments, the ketoreductase polypeptide with the specifiedamino acid residues at residue 96 can comprise one or more of thefeatures selected from: residue 49 is a polar amino acid residue otherthan K; residue 53 is an acidic amino acid residue; residue 54 is asmall or aliphatic amino acid residue other than T/P; residue 60 is analiphatic amino acid residue other than V; residue 95 is an aliphaticamino acid other than V; residue 97 is a small amino acid or G; residue109 is a basic amino acid residue other than K; residue 147 is analiphatic amino acid residue; residue 165 is a hydroxyl or small aminoacid residue; residue 197 is a small amino acid residue or G; residue223 is an aliphatic amino acid residue other than L; and residue 233 isa small amino acid residue or G.

In some embodiments, the ketoreductase polypeptide capable of convertingthe compound1-[4-(4-fluoro2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-propan-2-oneto the corresponding product (R)1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-propan-2-olis selected from SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, and 118.

As noted above, the recombinant ketoreductase polypeptides are alsocapable of reducing or converting acetophenone to the correspondingproduct (R)-1-phenylethanol. Additional polypeptides capable ofconverting acetophenone to the corresponding product (R) 1-phenylethanolinclude recombinant ketoreductase polypeptides comprising an amino acidsequence having a G, I, C or an aromatic amino acid at the amino acidresidue corresponding to residue 96 of SEQ ID NO:2 or SEQ ID NO:4.

In some embodiments, these ketoreductase polypeptides can comprise anamino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2or SEQ ID NO:4 with the proviso that the residue corresponding toresidue 96 is G, I, C or an aromatic amino acid. These engineeredketoreductase may optionally include one or more conservative mutationsat other residue positions within the polypeptide sequence.

In some embodiments, the ketoreductase polypeptides capable of reducingor converting acetophenone to the corresponding product(R)-1-phenylethanol can comprise a region having an amino acid sequencethat is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to a region or domain thereof ofSEQ ID NO:2 or SEQ ID NO:4, such as residues 90-233, with the provisothat the residue corresponding to residue 96 is G, I, C or an aromaticamino acid. In some embodiments of these ketoreductase polypeptides, oneor more of the remaining residues corresponding to residues 90-233 ofSEQ ID NO:2 or SEQ ID NO:4 may have a conservative substitution.

In some embodiments, the ketoreductase polypeptide comprises an aminoacid sequence having at least 90% amino acid sequence identity to SEQ IDNO:2 or SEQ ID NO:4 and comprise a G, I, C or an aromatic amino acid atthe amino acid residue corresponding to residue 96 of SEQ ID NO:2 or SEQID NO:4.

In some embodiments, the ketoreductase polypeptide capable of reducingor converting acetophenone to the corresponding product(R)-1-phenylethanol can comprise, in addition to the G, I, C or anaromatic amino acid at the amino acid residue corresponding to residue96, one or more of the features selected from: residue 94 is an aromaticamino acid residue or G; residue 153 is an aliphatic amino acid residueother than L; residue 199 is any amino acid residue other than L;residue 202 is an aliphatic amino acid residue other than A; and residue206 is an aromatic amino acid residue.

In some embodiments, the ketoreductase polypeptides capable of reducingor converting acetophenone to the corresponding product(R)-1-phenylethanol can comprise additionally one or more of thefeatures selected from: residue 49 is a polar amino acid residue otherthan K; residue 53 is an acidic amino acid residue; residue 54 is asmall or aliphatic amino acid residue other than T/P; residue 60 is analiphatic amino acid residue other than V; residue 95 is an aliphaticamino acid other than V; residue 97 is a small amino acid or G; residue109 is a basic amino acid residue other than K; residue 147 is analiphatic amino acid residue; residue 165 is a hydroxyl or small aminoacid residue; residue 197 is a small amino acid residue or G; residue223 is an aliphatic amino acid residue other than L; and residue 233 isa small amino acid residue or G.

In some embodiments, the ketoreductase polypeptide capable of reducingor converting acetophenone to the corresponding product(R)-1-phenylethanol is selected from SEQ ID NO: 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, and118. In some embodiments, the ketoreductase polypeptide capable ofreducing or converting acetophenone to the corresponding product(R)-1-phenylethanol is selected from SEQ ID NO: 120, 122, 124, 126, 128,130, 132, 134, 136, and 138.

In another aspect, the present disclosure provides polynucleotidesencoding the engineered ketoreductases described herein orpolynucleotides that hybridize to such polynucleotides under highlystringent conditions. The polynucleotide can include promoters and otherregulatory elements useful for expression of the encoded engineeredketoreductase, and can utilize codons optimized for specific desiredexpression systems. Exemplary polynucleotides include SEQ ID NO: 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, and 137.

In still another aspect, the present disclosure provides host cellscomprising the polynucleotides and/or expression vectors describedherein. The host cells may be Lactobacillus kefir or Lactobacillusbrevis, or they may be a different organism. The host cells can be usedfor the expression and isolation of the engineered ketoreductaseenzymes, or, alternatively, they can be used directly for the conversionof the keto substrate to the chiral alcohol product.

As will be appreciated by skilled artisans, the reduction reactionillustrated above generally requires a cofactor, which is normally NADHor NADPH, and can include a system for regenerating the cofactor, forexample glucose and glucose dehydrogenase. In some embodiments employingpurified engineered ketoreductase enzyme(s), such cofactors andoptionally such cofactor regeneration systems, will typically be addedto the reaction medium along with the substrate and the ketoreductaseenzyme(s). Like the engineered ketoreductase enzyme, any enzyme(s)comprising the cofactor regeneration system can be supplied to thereaction mixture in the form of extracts or lysates of such cells, or aspurified enzyme(s). In embodiments employing cell extracts or celllysates, the cells used to generate the extracts or lysates can beengineered to express the enzyme(s) comprising the cofactor regenerationsystems alone, or together with the engineered ketoreductase enzyme. Inembodiments employing whole cells, the cells can be engineered toexpress the enzyme(s) comprising the cofactor regeneration systems andthe engineered ketoreductase enzyme together.

Whether carrying out the method with whole cells, cell extracts orpurified ketoreductase enzymes, a single ketoreductase enzyme may beused or, alternatively, mixtures of two or more ketoreductase enzymesmay be used.

In various embodiments, the engineered enzymes can carry out thereduction or conversion reaction with a degree of enantioselectivity of≧99%. Thus, the above reactions can be used as a standard reaction forassessing the activity of the engineered ketoreductase enzymes ascompared to a reference ketoreductase, such as the ketoreductases of SEQID NO:2 or SEQ ID NO:4.

In some embodiments, because the engineered ketoreductase enzymesdescribed herein are highly stereoselective, the resultant product ofstructural formula (II) (“Compound (II)”) or structural formula (IV)(“Compound (IV)”) can be recovered in substantially stereochemicallypure form without the need to chirally separate it from thecorresponding enantiomer.

5. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the role of ketoreductases in the conversion of adefined substrate of Compound (I) to the chiral alcohol product ofCompound (II). The figure also shows use of a cofactor regeneratingsystem involving glucose dehydrogenase (GDH) and glucose.

FIG. 2 illustrates the role of ketoreductases in the conversion of adefined substrate of Compound (III) to the chiral alcohol product ofCompound (IV). The figure also shows use of a cofactor regeneratingsystem involving glucose dehydrogenase (GDH) and glucose.

6. DETAILED DESCRIPTION

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contextclearly indicates otherwise. Thus, for example, reference to “a protein”includes more than one protein, and reference to “a compound” refers tomore than one compound. In addition, the use of “or” means “and/or”unless stated otherwise. Similarly, “comprise,” “comprises,”“comprising” “include,” “includes,” and “including” are interchangeableand not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of”

The section headings used herein are for organizational purposes onlyand not to be construed as limiting the subject matter described.

6.1 DEFINITIONS

As used herein, the following terms are intended to have the followingmeanings.

“Ketoreductase” and “KRED” are used interchangeably herein to refer to apolypeptide that is capable of reducing a keto group to itscorresponding alcohol. More specifically, the ketoreductase polypeptidesof the present disclosure are capable of stereoselectively reducing thecompound of formula (I), supra to the alcohol product of formula (II),supra (see FIG. 1) and/or the compound of formula (III), supra to thealcohol product of formula (IV) (see FIG. 2). The polypeptide typicallyutilizes a cofactor reduced nicotinamide adenine dinucleotide (NADH) orreduced nicotinamide adenine dinucleotide phosphate (NADPH) as thereducing agent. Ketoreductases as used herein include naturallyoccurring (wild type) ketoreductases as well as non-naturally occurringengineered polypeptides generated by human manipulation (i.e.,recombinant polypeptides).

“Coding sequence” refers to that portion of a nucleic acid (e.g., agene) that encodes an amino acid sequence of a protein.

“Naturally-occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by human manipulation.

“Recombinant” when used with reference to, e.g., a cell, nucleic acid,or polypeptide, refers to a material, or a material corresponding to thenatural or native form of the material, that has been modified in amanner that would not otherwise exist in nature, or is identical theretobut produced or derived from synthetic materials and/or by manipulationusing recombinant techniques. Non-limiting examples include, amongothers, recombinant cells expressing genes that are not found within thenative (non-recombinant) form of the cell or express native genes thatare otherwise expressed at a different level.

“Percentage of sequence identity” and “percentage homology” are usedinterchangeably herein to refer to comparisons among polynucleotides andpolypeptides, and are determined by comparing two optimally alignedsequences over a comparison window, wherein the portion of thepolynucleotide or polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage may be calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Alternatively, the percentage may be calculated by determiningthe number of positions at which either the identical nucleic acid baseor amino acid residue occurs in both sequences or a nucleic acid base oramino acid residue is aligned with a gap to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison and multiplying the result by100 to yield the percentage of sequence identity. Those of skill in theart appreciate that there are many established algorithms available toalign two sequences. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith andWaterman, 1981, Adv. Appl. Math. 2:482, by the homology alignmentalgorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by thesearch for similarity method of Pearson and Lipman, 1988, Proc. Natl.Acad. Sci. USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG WisconsinSoftware Package), or by visual inspection (see generally, CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples ofalgorithms that are suitable for determining percent sequence identityand sequence similarity are the BLAST and BLAST 2.0 algorithms, whichare described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410 andAltschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website. This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas, the neighborhood word score threshold (Altschul et al, supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Exemplarydetermination of sequence alignment and % sequence identity can employthe BESTFIT or GAP programs in the GCG Wisconsin Software package(Accelrys, Madison Wis.), using default parameters provided.

“Reference sequence” refers to a defined sequence used as a basis for asequence comparison. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (I)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (II) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptide aretypically performed by comparing sequences of the two polynucleotidesover a “comparison window” to identify and compare local regions ofsequence similarity.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

“Substantial identity” refers to a polynucleotide or polypeptidesequence that has at least 80 percent sequence identity, at least 85percent identity and 89 to 95 percent sequence identity, more usually atleast 98 percent sequence identity, at least 99 percent sequenceidentity or at least 99.5 percent or more sequence identity as comparedto a reference sequence over a comparison window of at least 20 residuepositions, frequently over a window of at least 30-50 residues, whereinthe percentage of sequence identity is calculated by comparing thereference sequence to a sequence that includes deletions or additionswhich total 20 percent or less of the reference sequence over the windowof comparison. In specific embodiments applied to polypeptides, the term“substantial identity” means that two polypeptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights, share at least 80 percent sequence identity, preferably atleast 89 percent sequence identity, at least 95 percent sequenceidentity, at least 98% sequence identity, or at least 99 percentsequence identity. Preferably, residue positions which are not identicaldiffer by conservative amino acid substitutions.

“Corresponding to”, “reference to” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredketoreductase, can be aligned to a reference sequence by introducinggaps to optimize residue matches between the two sequences. In thesecases, although the gaps are present, the numbering of the residue inthe given amino acid or polynucleotide sequence is made with respect tothe reference sequence to which it has been aligned.

“Stereoselectivity” refers to the preferential formation in a chemicalor enzymatic reaction of one stereoisomer over another.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly reported in the art (typically as a percentage) asthe enantiomeric excess calculated therefrom according to the formula[major enantiomer−minor enantiomer]/[major enantiomer+minor enantiomer].Where the stereoisomers are diastereoisomers, the stereoselectivity isreferred to as diastereoselectivity, the fraction (typically reported asa percentage) of one diastereomer in the sum with others.

“Highly stereoselective” refers to a ketoreductase polypeptide that iscapable of converting or reducing the substrate to the correspondingproduct having the chemical formula (II) with at least about 85%stereomeric excess.

“Stereospecificity” refers to the preferential conversion in a chemicalor enzymatic reaction of one stereoisomer over another.Stereospecificity can be partial, where the conversion of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is converted.

“Improved enzyme property” refers to a ketoreductase polypeptide thatexhibits an improvement in any enzyme property as compared to areference ketoreductase. For the engineered ketoreductase polypeptidesdescribed herein, the comparison is generally made to the wild-typeketoreductase enzyme, although in some embodiments, the referenceketoreductase can be another improved engineered ketoreductase. Enzymeproperties for which improvement is desirable include, but are notlimited to, enzymatic activity (which can be expressed in terms ofpercent conversion of the substrate), thermal stability, pH activityprofile, cofactor requirements, refractoriness to inhibitors (e.g.,product inhibition), stereospecificity, and stereoselectivity (includingenantioselectivity).

“Increased enzymatic activity” refers to an improved property of theengineered ketoreductase polypeptides, which can be represented by anincrease in specific activity (e.g., product produced/time/weightprotein) or an increase in percent conversion of the substrate to theproduct (e.g., percent conversion of starting amount of substrate toproduct in a specified time period using a specified amount of KRED) ascompared to the reference ketoreductase enzyme. Exemplary methods todetermine enzyme activity are provided in the Examples. Any propertyrelating to enzyme activity may be affected, including the classicalenzyme properties of K_(m), V_(max) or k_(cat), changes of which canlead to increased enzymatic activity. Improvements in enzyme activitycan be from about 1.5 times the enzymatic activity of the correspondingwild-type ketoreductase enzyme, to as much as 2 times, 3 times, 4 times,5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times, ormore enzymatic activity than the naturally occurring ketoreductase oranother engineered ketoreductase from which the ketoreductasepolypeptides were derived. In specific embodiments, the engineeredketoreductase enzyme can exhibits improved enzymatic activity in therange of 1.5 to 50 times, 1.5 to 100 times greater than that of theparent ketoreductase enzyme. It is understood by the skilled artisanthat the activity of any enzyme is diffusion limited such that thecatalytic turnover rate cannot exceed the diffusion rate of thesubstrate, including any required cofactors. The theoretical maximum ofthe diffusion limit, or k_(cat)/K_(m), is generally about 10⁸ to 10⁹(M⁻¹ s⁻¹). Hence, any improvements in the enzyme activity of theketoreductase will have an upper limit related to the diffusion rate ofthe substrates acted on by the ketoreductase enzyme. Ketoreductaseactivity can be measured by any one of standard assays used formeasuring ketoreductase, such as a decrease in absorbance orfluorescence of NADPH due to its oxidation with the concomitantreduction of a ketone to an alcohol, or by product produced in a coupledassay. Comparisons of enzyme activities are made using a definedpreparation of enzyme, a defined assay under a set condition, and one ormore defined substrates, as further described in detail herein.Generally, when lysates are compared, the numbers of cells and theamount of protein assayed are determined as well as use of identicalexpression systems and identical host cells to minimize variations inamount of enzyme produced by the host cells and present in the lysates.

“Conversion” refers to the enzymatic conversion of the substrate to thecorresponding product. “Percent conversion” refers to the percent of thesubstrate that is reduced to the product within a period of time underspecified conditions. Thus, the “enzymatic activity” or “activity” of aketoreductase polypeptide can be expressed as “percent conversion” ofthe substrate to the product.

“Thermostable” refers to a ketoreductase polypeptide that maintainssimilar activity (more than 60% to 80% or more, for example) afterexposure to elevated temperatures (e.g., 40-80° C.) for a period of time(e.g., 0.5-24 hrs) compared to the untreated enzyme.

“Solvent stable” refers to a ketoreductase polypeptide that maintainssimilar activity (more than, e.g., 60% to 80%) after exposure to varyingconcentraions (e.g., 5-99%) of a solvent (e.g., isopropyl alcohol,tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene,butylacetate, methyl tert-butylether, etc.) or solvent mixture, for aperiod of time (e.g., 0.5-24 hrs) compared to the untreated enzyme.

“pH stable” refers to a ketoreductase polypeptide that maintains similaractivity (more than, e.g., 60% to 80%) after exposure to high or low pH(e.g., 4.5-6 or 8 to 12) for a period of time (e.g., 0.5-24 hrs)compared to the untreated enzyme.

“Thermo- and solvent stable” refers to a ketoreductase polypeptide thatare both thermostable and solvent stable.

“Derived from” as used herein in the context of engineered ketoreductaseenzymes, identifies the originating ketoreductase enzyme, and/or thegene encoding such ketoreductase enzyme, upon which the engineering wasbased. For example, the engineered ketoreductase enzyme of SEQ ID NO: 10was obtained by artificially evolving, over multiple generations thegene encoding the Lactobacillus kefir ketoreductase enzyme of SEQ IDNO:2. Thus, this engineered ketoreductase enzyme is “derived from” thewild-type ketoreductase of SEQ ID NO: 2.

“Hydrophilic Amino Acid or Residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of less than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophilicamino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn(N), L-Gln (Q), L-Asp (D), L-Lys (K) and L-Arg (R).

“Acidic Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of less than about 6when the amino acid is included in a peptide or polypeptide. Acidicamino acids typically have negatively charged side chains atphysiological pH due to loss of a hydrogen ion. Genetically encodedacidic amino acids include L-Glu (E) and L-Asp (D).

“Basic Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of greater than about6 when the amino acid is included in a peptide or polypeptide. Basicamino acids typically have positively charged side chains atphysiological pH due to association with hydronium ion. Geneticallyencoded basic amino acids include L-Arg (R) and L-Lys (K).

“Polar Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain that is uncharged at physiological pH, butwhich has at least one bond in which the pair of electrons shared incommon by two atoms is held more closely by one of the atoms.Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q),L-Ser (S) and L-Thr (T).

“Hydrophobic Amino Acid or Residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of greater than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophobicamino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu(L), L-Trp (W), L-Met (M), L-Ala (A) and L-Tyr (Y).

“Aromatic Amino Acid or Residue” refers to a hydrophilic or hydrophobicamino acid or residue having a side chain that includes at least onearomatic or heteroaromatic ring. Genetically encoded aromatic aminoacids include L-Phe (F), L-Tyr (Y) and L-Trp (W). Although owing to thepKa of its heteroaromatic nitrogen atom L-His (H) it is sometimesclassified as a basic residue, or as an aromatic residue as its sidechain includes a heteroaromatic ring, herein histidine is classified asa hydrophilic residue or as a “constrained residue” (see below).

“Constrained amino acid or residue” refers to an amino acid or residuethat has a constrained geometry. Herein, constrained residues includeL-pro (P) and L-his (H). Histidine has a constrained geometry because ithas a relatively small imidazole ring. Proline has a constrainedgeometry because it also has a five membered ring.

“Non-polar Amino Acid or Residue” refers to a hydrophobic amino acid orresidue having a side chain that is uncharged at physiological pH andwhich has bonds in which the pair of electrons shared in common by twoatoms is generally held equally by each of the two atoms (i.e., the sidechain is not polar). Genetically encoded non-polar amino acids includeL-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M) and L-Ala (A).

“Aliphatic Amino Acid or Residue” refers to a hydrophobic amino acid orresidue having an aliphatic hydrocarbon side chain. Genetically encodedaliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L) and L-Ile(I).

“Cysteine”. The amino acid L-Cys (C) is unusual in that it can formdisulfide bridges with other L-Cys (C) amino acids or other sulfanyl- orsulfhydryl-containing amino acids. The “cysteine-like residues” includecysteine and other amino acids that contain sulfhydryl moieties that areavailable for formation of disulfide bridges. The ability of L-Cys (C)(and other amino acids with —SH containing side chains) to exist in apeptide in either the reduced free —SH or oxidized disulfide-bridgedform affects whether L-Cys (C) contributes net hydrophobic orhydrophilic character to a peptide. While L-Cys (C) exhibits ahydrophobicity of 0.29 according to the normalized consensus scale ofEisenberg (Eisenberg et al., 1984, supra), it is to be understood thatfor purposes of the present disclosure L-Cys (C) is categorized into itsown unique group.

“Small Amino Acid or Residue” refers to an amino acid or residue havinga side chain that is composed of a total three or fewer carbon and/orheteroatoms (excluding the α-carbon and hydrogens). The small aminoacids or residues may be further categorized as aliphatic, non-polar,polar or acidic small amino acids or residues, in accordance with theabove definitions. Genetically-encoded small amino acids include L-Ala(A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) and L-Asp(D).

“Hydroxyl-containing Amino Acid or Residue” refers to an amino acidcontaining a hydroxyl (—OH) moiety. Genetically-encodedhydroxyl-containing amino acids include L-Ser (S) L-Thr (T) and L-Tyr(Y).

“Conservative” amino acid substitutions or mutations refer to theinterchangeability of residues having similar side chains, and thustypically involves substitution of the amino acid in the polypeptidewith amino acids within the same or similar defined class of aminoacids. However, as used herein, conservative mutations do not includesubstitutions from a hydrophilic to hydrophilic, hydrophobic tohydrophobic, hydroxyl-containing to hydroxyl-containing, or small tosmall residue, if the conservative mutation can instead be asubstitution from an aliphatic to an aliphatic, non-polar to non-polar,polar to polar, acidic to acidic, basic to basic, aromatic to aromatic,or constrained to constrained residue. Further, as used herein, A, V, L,or I can be conservatively mutated to either another aliphatic residueor to another non-polar residue. The table below shows exemplaryconservative substitutions.

Residue Possible Conservative Mutations A, L, V, I Other aliphatic (A,L, V, I) Other non-polar (A, L, V, I, G, M) G, M Other non-polar (A, L,V, I, G, M) D, E Other acidic (D, E) K, R Other basic (K, R) P, H Otherconstrained (P, H) N, Q, S, T Other polar Y, W, F Other aromatic (Y, W,F) C None

“Non-conservative substitution” refers to substitution or mutation of anamino acid in the polypeptide with an amino acid with significantlydiffering side chain properties. Non-conservative substitutions may useamino acids between, rather than within, the defined groups listedabove. In one embodiment, a non-conservative mutation affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine) (b) the charge or hydrophobicity, or (c) the bulkof the side chain.

“Deletion” refers to modification to the polypeptide by removal of oneor more amino acids to a reference polypeptide. Deletions can compriseremoval of 1, 2, 3, 4, 5 or more amino acids, 10 or more amino acids, 15or more amino acids, or 20 or more amino acids, up to 10% of the totalnumber of amino acids, or up to 20% of the total number of amino acidsmaking up the reference enzyme while retaining enzymatic activity and/orretaining the improved properties of an engineered ketoreductase enzyme.Deletions can be directed to the internal portions and/or terminalportions of the polypeptide. In various embodiments, the deletion cancomprise a continuous segment or can be discontinuous.

“Insertion” refers to modification to the polypeptide by addition of oneor more amino acids from the reference polypeptide. In some embodiments,the improved engineered ketoreductase enzymes comprise insertions of oneor more amino acids to the naturally occurring ketoreductase polypeptideas well as insertions of one or more amino acids to other engineeredketoreductase polypeptides. Insertions can be in the internal portionsof the polypeptide, or to the carboxy or amino terminus. Insertions asused herein include fusion proteins as is known in the art. Theinsertion can be a contiguous segment of amino acids or separated by oneor more of the amino acids in the naturally occurring polypeptide.

“Different from” or “differs from” with respect to a designatedreference sequence refers to difference of a given amino acid orpolynucleotide sequence when aligned to the reference sequence.Generally, the differences can be determined when the two sequences areoptimally aligned. Differences include insertions, deletions, orsubstitutions of amino acid residues in comparison to the referencesequence.

“Fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can be at least 14 amino acids long, at least 20amino acids long, at least 50 amino acids long or longer, and up to 70%,80%, 90%, 95%, 98%, and 99% of the full-length reference sequence, suchas a wild-type (SEQ ID NO:2 or SEQ ID NO:4) or engineered ketoreductasepolypeptide.

“Isolated polypeptide” refers to a polypeptide which is substantiallyseparated from other contaminants that naturally accompany it, e.g.,protein, lipids, and polynucleotides. The term embraces polypeptideswhich have been removed or purified from their naturally-occurringenvironment or expression system (e.g., host cell or in vitrosynthesis). The improved ketoreductase enzymes may be present within acell, present in the cellular medium, or prepared in various forms, suchas lysates or isolated preparations. As such, in some embodiments, theimproved ketoreductase enzyme can be an isolated polypeptide.

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (i.e., on a molaror weight basis it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure ketoreductase composition willcomprise about 60% or more, about 70% or more, about 80% or more, about90% or more, about 95% or more, and about 98% or more of allmacromolecular species by mole or % weight present in the composition.In some embodiments, the object species is purified to essentialhomogeneity (i.e., contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species. Solventspecies, small molecules (<500 Daltons), and elemental ion species arenot considered macromolecular species. In some embodiments, the isolatedimproved ketoreductases polypeptide is a substantially pure polypeptidecomposition.

“Stringent hybridization” is used herein to refer to conditions underwhich nucleic acid hybrids are stable. As known to those of skill in theart, the stability of hybrids is reflected in the melting temperature(T_(m)) of the hybrids. In general, the stability of a hybrid is afunction of ion strength, temperature, G/C content, and the presence ofchaotropic agents. The T_(m) values for polynucleotides can becalculated using known methods for predicting melting temperatures (see,e.g., Baldino et al., Methods Enzymology 168:761-777; Bolton et al.,1962, Proc. Natl. Acad. Sci. USA 48:1390; Bresslauer et al., 1986, Proc.Natl. Acad. Sci. USA 83:8893-8897; Freier et al., 1986, Proc. Natl.Acad. Sci. USA 83:9373-9377; Kierzek et al., Biochemistry 25:7840-7846;Rychlik et al., 1990, Nucleic Acids Res 18:6409-6412 (erratum, 1991,Nucleic Acids Res 19:698); Sambrook et al., supra); Suggs et al., 1981,In Developmental Biology Using Purified Genes (Brown et al., eds.), pp.683-693, Academic Press; and Wetmur, 1991, Crit. Rev Biochem Mol Biol26:227-259. All publications incorporate herein by reference). In someembodiments, the polynucleotide encodes the polypeptide disclosed hereinand hybridizes under defined conditions, such as moderately stringent orhighly stringent conditions, to the complement of a sequence encoding anengineered ketoreductase enzyme of the present disclosure.

“Hybridization stringency” relates to such washing conditions of nucleicacids. Generally, hybridization reactions are performed under conditionsof lower stringency, followed by washes of varying but higherstringency. The term “moderately stringent hybridization” refers toconditions that permit target-polynucleotides to bind a complementarypolynucleotide that has about 60% sequence identity, about 75% sequenceidentity, about 85% sequence identity; about 90% sequence identity, orwith about 95% or greater sequence identity to thetarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C.

“High stringency hybridization” refers generally to conditions that areabout 10° C. or less from the thermal melting temperature T_(m) asdetermined under the solution condition for a defined polynucleotidesequence. In some embodiments, a high stringency condition refers toconditions that permit hybridization of only those nucleic acidsequences that form stable hybrids in 0.018M NaCl at 65° C. (i.e., if ahybrid is not stable in 0.018M NaCl at 65° C., it will not be stableunder high stringency conditions, as contemplated herein). Highstringency conditions can be provided, for example, by hybridization inconditions equivalent to 50% formamide, 5× Denhart's solution, 5×SSPE,0.2% SDS at 42° C., followed by washing in 0.1×SSPE, and 0.1% SDS at 65°C. Other high stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

“Heterologous” polynucleotide refers to any polynucleotide that isintroduced into a host cell by laboratory techniques, and includespolynucleotides that are removed from a host cell, subjected tolaboratory manipulation, and then reintroduced into a host cell.

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is efficiently expressed in the organismof interest. Although the genetic code is degenerate in that most aminoacids are represented by several codons, called “synonyms” or“synonymous” codons, it is well known that codon usage by particularorganisms is nonrandom and biased towards particular codon triplets.This codon usage bias may be higher in reference to a given gene, genesof common function or ancestral origin, highly expressed proteins versuslow copy number proteins, and the aggregate protein coding regions of anorganism's genome. In some embodiments, the polynucleotides encoding theketoreductases enzymes may be codon optimized for optimal productionfrom the host organism selected for expression.

“Preferred, optimal, high codon usage bias codons” refersinterchangeably to codons that are used at higher frequency in theprotein coding regions than other codons that code for the same aminoacid. The preferred codons may be determined in relation to codon usagein a single gene, a set of genes of common function or origin, highlyexpressed genes, the codon frequency in the aggregate protein codingregions of the whole organism, codon frequency in the aggregate proteincoding regions of related organisms, or combinations thereof. Codonswhose frequency increases with the level of gene expression aretypically optimal codons for expression. A variety of methods are knownfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariat analysis, for example, using cluster analysis orcorrespondence analysis, and the effective number of codons used in agene (see GCG CodonPreference, Genetics Computer Group WisconsinPackage; CodonW, John Peden, University of Nottingham; McInerney, J. O,1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res.222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables areavailable for a growing list of organisms (see for example, Wada et al.,1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl.Acids Res. 28:292; Duret, et al., supra; Henaut and Danchin,“Escherichia coli and Salmonella,” 1996, Neidhardt, et al. Eds., ASMPress, Washington D.C., p. 2047-2066. The data source for obtainingcodon usage may rely on any available nucleotide sequence capable ofcoding for a protein. These data sets include nucleic acid sequencesactually known to encode expressed proteins (e.g., complete proteincoding sequences-CDS), expressed sequence tags (ESTS), or predictedcoding regions of genomic sequences (see for example, Mount, D.,Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E.C., 1996, Methods Enzymol. 266:259-281; Tiwari et al., 1997, Comput.Appl. Biosci. 13:263-270).

“Control sequence” is defined herein to include all components, whichare necessary or advantageous for the expression of a polypeptide of thepresent disclosure. Each control sequence may be native or foreign tothe nucleic acid sequence encoding the polypeptide. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleic acid sequenceencoding a polypeptide.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed at a position relative to thecoding sequence of a polynucleotide sequence such that the controlsequence directs or affects the expression of a polynucleotide and/orpolypeptide encoded by the polynucleotide.

“Promoter sequence” is a nucleic acid sequence that is recognized by ahost cell for expression of a polynucleotide, such as a polynucleotidecontaining the coding region. Generally, the promoter sequence containstranscriptional control sequences, which mediate expression of thepolynucleotide. The promoter may be any nucleic acid sequence whichshows transcriptional activity in the host cell of choice includingmutant, truncated, and hybrid promoters, and may be obtained from genesencoding extracellular or intracellular polypeptides either homologousor heterologous to the host cell.

“Cofactor regeneration system” refers to a set of reactants thatparticipate in a reaction that reduces the oxidized form of the cofactor(e.g., NADP⁺ to NADPH). Cofactors oxidized by theketoreductase-catalyzed reduction of the keto substrate are regeneratedin reduced form by the cofactor regeneration system. Cofactorregeneration systems comprise a stoichiometric reductant that is asource of reducing hydrogen equivalents and is capable of reducing theoxidized form of the cofactor. The cofactor regeneration system mayfurther comprise a catalyst, for example an enzyme catalyst, thatcatalyzes the reduction of the oxidized form of the cofactor by thereductant. Cofactor regeneration systems to regenerate NADH or NADPHfrom NAD⁺ or NADP⁺, respectively, are known in the art and may be usedin the methods described herein.

6.2 KETOREDUCTASE ENZYMES

The present disclosure provides engineered or recombinant ketoreductase(“KRED”) enzymes that are capable of reducing or converting a definedketo substrate to its corresponding alcohol product and having animproved property when compared with the naturally-occurring, wild-typeketoreductase enzyme obtained from Lactobacillus kefir or Lactobacillusbrevis, or another reference ketoreductase enzyme, such as anotherengineered ketoreductase enzyme. In the embodiments herein, therecombinant ketoreductase polypeptides are capable of stereoselectivelyreducing or converting the substrate1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-one,as represented by the structure of formula (I), to the product(R)-1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-ol,as represented by the structure of formula (II), as further discussedbelow. The ketoreductase have an improved property in converting thesubstrate of formula (I) to the product of formula (II) as compared tothe naturally occurring ketoreductase enzymes of Lactobacillus kefir orLactobacillus bacillus.

In some embodiments, the recombinant ketoreductase polypeptides are alsocapable of reducing or converting the substrate acetophenone, asrepresented by structural formula (III) to the chiral alcohol product(R) 1-phenylethanol, as represented by the structure of formula (IV), asfurther discussed below. Thus, the engineered ketoreductases can becompared to reference ketoreductases using the substrate1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-oneor acetophenone, or both. In some embodiments, one reference substrate(e.g.,1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-one)can be substituted with the other reference substrate (e.g.,acetophenone) when comparing the activity of the engineeredketoreductases to the reference ketoreductases (e.g., SEQ ID NO:2 or SEQID NO:4).

In the embodiments herein, the ketoreductase polypeptides comprise atleast (1) an amino acid residue at position 94 which is an aromaticamino acid residue or G, and/or (2) an amino acid residue at position 96which is an amino acid other than S/N in the corresponding residueposition of the wild-type L. kefir or L. brevis sequences of SEQ ID NO:2and 4, respectively. Thus, in various embodiments herein, theketoreductases of the present disclosure comprise at least the followingamino acid substitutions: (1) residue 94 is modified from A→an aromaticamino acid residue or G, and/or (2) residue 96 is modified from S/N→anyamino acid other than S/N, for a polypeptide corresponding to the aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4.

These non-naturally occurring ketoreductases can be generated by variouswell-known techniques, such as in vitro mutagenesis or directedevolution of the genetic material encoding the ketoreductase enzyme ofLactobacillus kefir or Lactobacillus brevis, and identifyingpolynucleotides that express engineered enzymes with a desired property.The capabilities of the KREDs can refer to their properties as singlepolypeptides or to their properties in the form of multimers, as theymay be present in the wild-type enzyme.

Mutagenesis and directed evolution techniques useful for the purposesherein are amply described in the literature: Ling, et al., 1997,“Approaches to DNA mutagenesis: an overview,” Anal. Biochem.254(2):157-78; Dale et al., 1996, “Oligonucleotide-directed randommutagenesis using the phosphorothioate method,” Methods Mol. Biol.57:369-74; Smith, 1985, “In vitro mutagenesis,” Ann. Rev. Genet.19:423-462; Botstein et al., 1985, “Strategies and applications of invitro mutagenesis,” Science 229:1193-1201; Carter, 1986, “Site-directedmutagenesis,” Biochem. J. 237:1-7; Kramer et al., 1984, “Point MismatchRepair,” Cell, 38:879-887; Wells et al., 1985, “Cassette mutagenesis: anefficient method for generation of multiple mutations at defined sites,”Gene 34:315-323; Minshull et al., 1999, “Protein evolution by molecularbreeding,” Curr Opin Chem Biol 3:284-290; Christians et al., 1999,“Directed evolution of thymidine kinase for AZT phosphorylation usingDNA family shuffling,” Nature Biotech 17:259-264; Crameri et al., 1998,“DNA shuffling of a family of genes from diverse species acceleratesdirected evolution,” Nature 391:288-291; Crameri et al., 1997,“Molecular evolution of an arsenate detoxification pathway by DNAshuffling,” Nature Biotech 15:436-438; Zhang et al., 1997, “Directedevolution of an effective fructosidase from a galactosidase by DNAshuffling and screening,” Proc Natl Acad Sci USA 94:45-4-4509; Crameriet al., 1996, “Improved green fluorescent protein by molecular evolutionusing DNA shuffling,’ Nature Biotech 14:315-319; Stemmer, 1994, “Rapidevolution of a protein in vitro by DNA shuffling,” Nature 370:389-391;Stemmer, 1994, “DNA shuffling by random fragmentation and reassembly: Invitro recombination for molecular evolution,” Proc Natl Acad Sci USA91:10747-10751; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO00/42651; WO 01/75767 and U.S. Pat. No. 6,537,746. All publications areincorporated herein by reference.

The naturally occurring polynucleotide encoding the naturally occurringketoreductase of Lactobacillus kefir and Lactobacillus brevis (alsoreferred to as “alcohol dehydrogenase” or “ADH”) can be obtained fromthe isolated polynucleotide known to encode the ketoreductase activity(e.g., Genbank accession no. AAP94029 GI:33112056 for Lactobacilluskefir and Genbank accession no. CAD66648 GI:28400789 for Lactobacillusbrevis). Alternatively, a polynucleotide encoding the naturallyoccurring ketoreductases can be synthesized by polynucleotide synthesismethodologies known in the art based on the reported polynucleotidesequence of the ketoreductase-encoding gene. In various embodiments, asfurther described below, the naturally occurring polynucleotide encodingthe ketoreductase, as well as the engineered ketoreductases, can becodon optimized to the codons preferred by a specific host cell used forexpression of the enzyme.

The parent or reference polynucleotide encoding the naturally occurringor wild type ketoreductase is subjected to mutagenic processes, forexample random mutagenesis and recombination, to introduce mutationsinto the polynucleotide. The mutated polynucleotide is expressed andtranslated, thereby generating engineered ketoreductase enzymes withmodifications to the polypeptide. As used herein, “modifications”include amino acid substitutions, deletions, and insertions. Any one ora combination of modifications can be introduced into the naturallyoccurring enzymatically active polypeptide to generate engineeredenzymes, which are then screened by various methods to identifypolypeptides, and corresponding polynucleotides, having a desiredimprovement in a specific enzyme property. A polynucleotide encoding anengineered ketoreductase with an improved property can be subjected toadditional rounds of mutagenesis treatments to generate polypeptideswith further improvements in the desired enzyme property. Enzymeproperties for which improvement is desirable include, but are notlimited to, enzymatic activity, thermal stability, pH activity profile,cofactor requirements, refractoriness to inhibitors (e.g., productinhibition), sterospecificity, stereoselectivity, and solvent stability.

In some embodiments, the recombinant ketoreductase comprise engineeredpolypeptides derived from, and thus can be compared to, Lactobacilluskefir ketoreductase of SEQ ID NO:2 or Lactobacillus brevis ketoreductaseof SEQ ID NO:4. In the descriptions here, the amino acid residueposition is determined with respect to a reference polypeptide. For thereference sequences of SEQ ID NO:2 or SEQ ID NO:4, the numbering beginsfrom the initiating methionine (M) residue (i.e., M represents residueposition 1), although it will be understood by the skilled artisan thatthis initiating methionine residue may be removed by biologicalprocessing machinery, such as in a host cell or in vitro translationsystem, to generate a mature protein lacking the initiating methionineresidue. Where the amino acid residues at the same residue positiondiffer between the two reference ketoreductases, the different residuesare denoted by a “/” with the arrangement being “kefir residue/brevisresidue”. A substitution mutation, which is a replacement of an aminoacid residue in a reference sequence, for example the wildtypeketoreductases of SEQ ID NO:2 and SEQ ID NO:4, with a different aminoacid residue is denoted by the symbol “→”.

The number of modifications to a reference polypeptide, such as thenaturally occurring polypeptide of SEQ ID NO:2 or SEQ ID NO:4, thatproduces an improved ketoreductase property may comprise one or moreamino acids, 2 or more amino acids, 3 or more amino acids, 4 or moreamino acid, 5 or more amino acids, 6 or more amino acids, 8 or moreamino acids, 10 or more amino acids, or 15 or more amino acids, 20 ormore amino acids, up to 10% of the total number of amino acids, up to20% of the total number of amino acids, or up to 30% of the total numberof amino acids of the reference enzyme sequence. As such, thepolypeptides of the present disclosure can differ from the referencepolypeptide (e.g., SEQ ID NO:2 or SEQ ID NO:4) in 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 14 16, 18, 20 or 25 or more amino acids, or up to 5%of the amino acids, up to 10% of the amino acids, up to 20% of the aminoacids, or up to 30% of the total number of amino acids of the referencesequence.

In various embodiments, the modifications to the reference polypeptideto produce the improved enzyme property can comprise substitutions at 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14 16, 18, 20, 25, or more aminoacids, or up to 5% of the amino acids, up to 10% of the amino acids, upto 20% of the amino acids, or up to 30% of the total number of aminoacids of the reference sequence, such as SEQ ID NO:2 or SEQ ID NO:4. Thesubstitutions for generating an improved ketoreductase can compriseconservative substitutions, non-conservative substitutions, as well ascombinations of conservative and non-conservative substitutions.

In some embodiments, the ketoreductase polypeptides have increasedenzyme activity in stereoselectively reducing or converting thesubstrate1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-oneto the product(R)-1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-olas compared to the activity of a wild-type reference ketoreductases ofSEQ ID NO:2 or SEQ ID NO:4. In some embodiments, the increased enzymeactivity is at least 1.5 times or more the enzyme activity of thewild-type reference polypeptides. In some embodiments, the increasedenzyme activity is at least 2.0 times or more enzyme activity, at least3.0 times or more enzyme activity, at least 5 times or more enzymeactivity, at least 10 times or more enzyme activity, at least 20 timesor more enzyme activity, at least 25 times or more enzyme activity, atleast 50 times or more enzyme activity, at least 75 times or more enzymeactivity, at least 100 times or more enzyme activity as compared to theactivity of reference polypeptides SEQ ID NO:2 or SEQ ID NO:4.

In some embodiments, the ketoreductase polypeptides have an increasedconversion rate in stereoselectively reducing or converting1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-oneto the product(R)-1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-olas compared to the conversion rate of a wild-type referenceketoreductase of SEQ ID NO:2 or SEQ ID NO:4 under a defined condition.In some embodiments, the engineered ketoreductases are characterized bya conversion of greater than 70%, of greater than 80%, of greater than90%, of greater than 95%, of greater than 98% or of greater than 99% ofthe substrate under a defined condition. An exemplary defined conditionis conversion in 24 hours of 10 g/L substrate with about 10 g/L of theKRED.

In some embodiments, the ketoreductase polypeptides have an increasedenzyme activity in stereoselectively reducing or converting thesubstrate acetophenone to the product (R)-1-phenylethanol as compared tothe activity of a wild-type reference ketoreductases of SEQ ID NO:2 orSEQ ID NO:4. In some embodiments, the increased enzyme activity is atleast 1.5 times or more the enzyme activity of the wild-type referencepolypeptides. In some embodiments, the increased enzyme activity is atleast 2.0 times or more enzyme activity, at least 3.0 times or moreenzyme activity, at least 5 times or more enzyme activity, at least 10times or more enzyme activity, at least 20 times or more enzymeactivity, at least 25 times or more enzyme activity, at least 50 timesor more enzyme activity, at least 75 times or more enzyme activity, atleast 100 times or more enzyme activity as compared to the activity ofreference polypeptides SEQ ID NO:2 or SEQ ID NO:4.

In some embodiments, the ketoreductase polypeptides have an increasedconversion rate in stereoselectively reducing or converting thesubstrate acetophenone to the product (R)-1-phenylethanol as compared tothe activity of a wild-type reference ketoreductases of SEQ ID NO:2 orSEQ ID NO:4 under a defined condition. In some embodiments, theengineered ketoreductases are characterized by conversion of greaterthan 70%, of greater than 80%, of greater than 90%, of greater than 95%,of greater than 98% or of greater than 99% of the substrate under thedefined condition. An exemplary defined condition is conversion in 24hours of 10 g/L substrate with about 10 g/L of the KRED.

In various embodiments, the ketoreductase polypeptide is capable ofstereoselectively reducing or converting the substrate1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-oneto the product(R)-1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-olwith at least 1.5 times the activity of SEQ ID NO:2 or SEQ ID NO:4, andcomprises an amino acid sequence having (1) an aromatic amino acid or Gat the corresponding residue position 94 of the reference sequence SEQID NO:2 or SEQ ID NO:4, or (2) an amino acid other than S/N at thecorresponding residue position 96 of the reference sequence SEQ ID NO:2or SEQ ID NO:4. In some embodiments, the amino acid residue at thecorresponding residue position 96 is other than S and N. In some ofthese embodiments, the ketoreductase polypeptide can differ from thereference sequence in 2 or more amino acid residues, 3 or more aminoacid residues, or 4 or more amino acid residues as compared to thereference sequence, as discussed below.

In some embodiments, the ketoreductase polypeptide capable ofstereoselectively reducing or converting the substrate1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-oneto the product(R)-1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-olcomprises an amino acid sequence having an aromatic amino acid or G atthe corresponding residue position 94 of the reference sequence SEQ IDNO:2 or SEQ ID NO:4. As such, in some embodiments, the ketoreductasepolypeptide comprises a modification at residue 94 of A→an aromaticamino acid residue or G of the corresponding sequence of SEQ ID NO:2 orSEQ ID NO:4. In some of these embodiments, the ketoreductase polypeptidecan differ from the reference sequence in 2 or more amino acid residues,3 or more amino acid residues, or 4 or more amino acid residues ascompared to the reference sequence, as discussed below.

In some embodiments, the ketoreductase polypeptide comprises an aminoacid sequence having an amino acid F, W, H, or Y at the correspondingamino acid residue of residue 94 of SEQ ID NO:2 or SEQ ID NO:4. As such,in some embodiments, the ketoreductase polypeptide comprises amodification at residue 94 of A→F, W, H, or Y of the correspondingsequence of SEQ. ID NO:2 or SEQ ID NO:4. In some embodiments, the aminoacid at residue 94 is F.

In some embodiments, the ketoreductase polypeptides capable ofstereoselectively converting the substrate1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-oneto the product(R)-1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-olwith the specified amino acids at residue 94 can comprise modificationsat other amino acid residues of the corresponding SEQ ID NO:2 or SEQ IDNO:4, including non-conservative or conservative substitutions.Non-conservative substitutions can be at amino acid residuescorresponding to residue positions 53, 54, 96, 97, 147, 165, 153, 197,199, 206, 223, and 233, as further discussed below. Additionalsubstitutions, when present, can comprise one or more conservativesubstitutions at other amino acid residue positions.

Thus, in some embodiments, the recombinant ketoreductase polypeptidescomprise an amino acid sequence which differs in 1 to 25 amino acidpositions from SEQ ID NO: 2 with the proviso that the amino acid residuecorresponding to residue 94 of SEQ ID NO:2 or SEQ ID NO:4 is aromaticamino acid or G. As such, some polypeptides comprise an amino sequencewhich differs from SEQ ID NO: 2 or SEQ ID NO:4 in 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 14, 16, 18, 20, or 25 amino acid positions, forexample, in 1-25 amino acid positions, in 1-20 amino acid positions, in1-18 amino acid positions, in 1-16 amino acid positions, 1-14 amino acidpositions, in 1-12 amino acid positions, in 1-11 amino acid positions,in 1-10 amino acid positions, in 1-9 amino acid positions, in 1-8 aminoacid positions, in 1-7 amino acid positions, in 1-6 amino acidpositions, in 1-5 amino acid positions, in 1-4 amino acid positions, in1-3 amino acid positions, or in 1-2 amino acid positions.

In some embodiments, the ketoreductase polypeptides capable ofstereoselectively reducing or converting the substrate1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-oneto the product(R)-1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-olcomprises an amino acid sequence with an aromatic amino acid or G atresidue corresponding to residue 94 of SEQ ID NO:2 or SEQ ID NO:4 andone or more of the features selected from: (1) amino acid at residuecorresponding to residue 96 is any amino acid other than S/N; (2) aminoacid residue corresponding to residue 153 is an aliphatic amino acidother than L; (3) amino acid residue corresponding to residue 199 is anyamino acid other than L; (4) amino acid residue corresponding to residue202 is G or an aliphatic amino acid other than A; and (5) amino acidresidue corresponding to residue 206 is an aromatic amino acid. Thus, insome embodiments, the ketoreductase polypeptides can comprise an aminoacid sequence with modifications at residue 94 of A→an aromatic aminoacid residue or G and one or more modifications selected from: 96(S/N→any amino acid other than S/N); 153 (L→an aliphatic amino acidresidue other than L); 199 (L→any amino acid residue other than L); 202(A→G or an aliphatic amino acid residue other than A); and 206 (M→anaromatic amino acid residue) of the corresponding sequence of SEQ IDNO:2 or SEQ ID NO:4.

In some embodiments, the amino acid residues, and correspondingmutations, in the above residues are selected from: residue 153 is G orA; residue 199 is K, I, N, R, V, Q, or W; residue 202 is I, L, or G; andresidue 206 is F.

In some embodiments, the ketoreductase polypeptides capable ofstereoselectively reducing or converting the substrate1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-oneto the product(R)-1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-olcomprises an amino acid sequence with a G, F, Y, or I at the amino acidresidue corresponding to residue 96 of SEQ ID NO:2 or SEQ ID NO:4. Assuch, in some embodiments, the ketoreductase polypeptides comprise amodification at residue 96 of S/N→G, F, Y, or I of the correspondingsequence of SEQ. ID NO:2 or SEQ ID NO:4. In some of these embodiments,the ketoreductase polypeptide can differ from the reference sequence in2 or more amino acid residues, 3 or more amino acid residue, or 4 ormore amino acid residues as compared to the reference sequence, asdiscussed below.

In some embodiments, the ketoreductase polypeptides are also capable ofstereoselectively reducing or converting the substrate acetophenone tothe product (R)-1-phenylethanol. In some of these embodiments, theketoreductase polypeptide comprises an amino acid sequence having a G,I, C or an aromatic amino acid at the amino acid residue correspondingto residue 96 of SEQ ID NO:2 or SEQ ID NO:4. As such, in someembodiments, the ketoreductase polypeptides comprise a modification atresidue 96 of S/N→G, I, C or an aromatic amino acid residue of thecorresponding sequence of SEQ ID NO:2 or SEQ ID NO:4. In some of theseembodiments, the ketoreductase polypeptide can differ from the referencesequence in 2 or more amino acid residues, 3 or more amino acid residue,or 4 or more amino acid residues as compared to the reference sequence,as discussed below.

In some embodiments, the ketoreductase polypeptides with the specifiedamino acids at residue 96 can comprise modifications at other aminoacids residues of the corresponding SEQ ID NO:2 or SEQ ID NO:4,including non-conservative or conservative substitutions.Non-conservative substitutions can be at amino acid residuescorresponding to residue positions 53, 54, 96, 97, 147, 165, 153, 197,199, 206, 223, and 233, as further discussed below. Additionalsubstitutions, when present, can comprise one or more conservativesubstitutions at other amino acid residue positions.

In some embodiments, ketoreductase polypeptide comprises an amino acidsequence which differs in 1 to 25 amino acid positions from SEQ ID NO: 2with the proviso that the amino acid residue corresponding to residue 96of SEQ ID NO:2 or SEQ ID NO:4 is a G, F, Y, or I. As such, somepolypeptides comprise an amino sequence which differs from SEQ ID NO: 2or SEQ ID NO:4 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20,or 25 amino acid positions, for example, in 1-25 amino acid positions,in 1-20 amino acid positions, in 1-18 amino acid positions, in 1-16amino acid positions, 1-14 amino acid positions, in 1-12 amino acidpositions, in 1-11 amino acid positions, in 1-10 amino acid positions,in 1-9 amino acid positions, in 1-8 amino acid positions, in 1-7 aminoacid positions, in 1-6 amino acid positions, in 1-5 amino acidpositions, in 1-4 amino acid positions, in 1-3 amino acid positions, orin 1-2 amino acid positions.

In some embodiments, ketoreductase polypeptide comprises an amino acidsequence which differs in 1 to 25 amino acid positions from SEQ ID NO: 2and comprises G, I, C or an aromatic amino acid at the residuecorresponding to residue 96 of SEQ ID NO:2 or SEQ ID NO:4. As such, somepolypeptides comprise an amino sequence which differs from SEQ ID NO: 2or SEQ ID NO:4 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20,or 25 amino acid positions, for example, in 1-25 amino acid positions,in 1-20 amino acid positions, in 1-18 amino acid positions, in 1-16amino acid positions, 1-14 amino acid positions, in 1-12 amino acidpositions, in 1-11 amino acid positions, in 1-10 amino acid positions,in 1-9 amino acid positions, in 1-8 amino acid positions, in 1-7 aminoacid positions, in 1-6 amino acid positions, in 1-5 amino acidpositions, in 1-4 amino acid positions, in 1-3 amino acid positions, orin 1-2 amino acid positions.

In some embodiments, the ketoreductase polypeptide comprises an aminoacid sequence with the specified amino acid at residue 96 of SEQ ID NO:2or SEQ ID NO:4 above, and one or more of the following features: (1)amino acid at residue corresponding to residue 94 is an aromatic aminoacid or G; (2) amino acid residue corresponding to residue 153 is analiphatic amino acid other than L; (3) amino acid residue correspondingto residue 199 is any amino acid other than L; (4) amino acid residuecorresponding to residue 202 is G or an aliphatic amino acid other thanA; and (5) amino acid residue corresponding to residue 206 is anaromatic amino acid.

Thus, in some embodiments, the ketoreductase polypeptide comprises anamino acid sequence with modifications at residue 96 of S/N→G, F, Y, orI and one or more modifications at residue: 94 (A→an aromatic amino acidresidue or G); 153 (L→an aliphatic amino acid residue other than L); 199(L→any amino acid residue other than L); 202 (A→G or an aliphatic aminoacid residue other than A); and 206 (M→an aromatic amino acid residue)of the corresponding sequence of SEQ ID NO:2 or SEQ ID NO:4.

In some embodiments, the ketoreductase polypeptide comprises an aminoacid sequence with modifications at residue 96 of S/N→G, I, C and one ormore modifications at the following residues: 94 (A→an aromatic aminoacid residue or G); 153 (L→an aliphatic amino acid residue other thanL); 199 (L→any amino acid residue other than L); 202 (A→G or analiphatic amino acid residue other than A); and 206 (M→an aromatic aminoacid residue) of the corresponding sequence of SEQ ID NO:2 or SEQ IDNO:4.

In some embodiments, the amino acid residues, and correspondingmutations, in the above residues can be selected from one or more of thefollowing: residue 94 is F or G; residue 153 is G or A; residue 199 isK, I, N, R, V, Q, or W; residue 202 is I, L, or G; and residue 206 is F.

In some embodiments, additional mutations can be incorporated into allof the ketoreductase polypeptide embodiments above to enhance one ormore properties of the polypeptide activity, such as, among others,enzyme activity, thermal stability, and/or solvent stability. Thus, insome embodiments, the ketoreductase polypeptides can comprise, inaddition to all of embodiments above, one or more of the followingfeatures: (1) amino acid at residue corresponding to residue 49 is apolar amino acid residue other than K; (2) amino acid at residuecorresponding to residue 53 is an acidic amino acid residue; (3) aminoacid at residue corresponding to residue 54 is a small or aliphaticamino acid residue other than T/P; (4) amino acid at residuecorresponding to residue 60 is an aliphatic amino acid residue otherthan V; (5) amino acid at residue corresponding to residue 95 is analiphatic amino acid other than V; (6) amino acid at residuecorresponding to residue 97 is a small amino acid or G; (7) amino acidat residue corresponding to residue 109 is a basic amino acid residueother than K; (8) amino acid at residue corresponding to residue 147 isan aliphatic amino acid residue; (9) amino acid at residue correspondingto residue 165 is a hydroxyl or small amino acid residue; (10) aminoacid at residue corresponding to residue 197 is a small amino acidresidue or G; (11) amino acid at residue corresponding to residue 223 isan aliphatic amino acid residue other than L; and (12) amino acid atresidue corresponding to residue 233 is a small amino acid residue or G.

Thus, in these embodiments, the ketoreductase polypeptide can comprisean amino acid sequence with the specified modifications described forevery embodiment of the ketoreductases above and one or more of thefollowing modifications: 49 (K→a polar amino acid residue other than K);53 (G/T→an acidic amino acid residue); 54 (T/P→a small or aliphaticamino acid residue other than T/P); 60 (V/F→an aliphatic amino acidresidue other than V); 95 (V→an aliphatic amino acid other than V); 97(K→a small amino acid or G); 109 (K→a basic amino acid residue otherthan K); 147 (F→an aliphatic amino acid residue); 165 (I→a hydroxyl orsmall amino acid residue); 197 (D→a small amino acid residue or G; 223(I→an aliphatic amino acid residue other than L); and 233 (D/N→a smallamino acid residue or G) of the corresponding sequence of SEQ ID NO:2 orSEQ ID NO:4.

In some embodiments, the ketoreductase polypeptides comprises an aminoacid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2 orSEQ ID NO:4 with the proviso that the residues corresponding to (1)residue 94 is an aromatic amino acid residue or G, and/or (2) residue 96is an amino acid residue other than S/N. In some embodiments, the aminoacid residue at the corresponding residue position 96 is other than Sand N.

In some embodiments, the ketoreductase polypeptides can comprise anamino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2or SEQ ID NO:4 with the proviso that the residues corresponding to: (1)residue 94 is F, W, H, or Y; (2) residue 96 is G, F, Y, or I; or (3)residue 96 is G, I, C or an aromatic amino acid.

In some embodiments, the ketoreductase polypeptides of the invention cancomprise a region having an amino acid sequence that is at least about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to a region or domain thereof of SEQ ID NO:2 or SEQ IDNO:4, such as residues 90-233, with the proviso that the residuescorresponding to (1) residue 94 is an aromatic amino acid residue or G,and/or (2) residue 96 is an amino acid residue other than S/N of thecorresponding residue of SEQ ID NO:2 or SEQ ID NO:4. In someembodiments, the amino acid residue at the corresponding residueposition 96 is other than S and N. In some embodiments of theseketoreductase polypeptides, one or more of the remaining residuescorresponding to residues 90-233 of SEQ ID NO:2 or SEQ ID NO:4 may beconservatively mutated.

In some embodiments, the ketoreductase polypeptides can comprise aregion having an amino acid sequence that is at least about 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a region or domain thereof of SEQ ID NO:2 or SEQ ID NO:4,such as residues 90-233, with the proviso that the residuescorresponding to: (1) residue 94 is F, W, H, or Y; (2) residue 96 is G,F, Y, or I; or (3) residue 96 is G, I, C or an aromatic amino acid ofthe corresponding residue of SEQ ID NO:2 or SEQ ID NO:4. In someembodiments of these ketoreductase polypeptides, one or more of theremaining residues corresponding to residues 90-233 of SEQ ID NO:2 orSEQ ID NO:4 may be conservatively mutated.

In some embodiments, the engineered ketoreductase polypeptide isselected from the amino acid sequences recited in Table 1. Table 1 ranksthe enzyme activity measured for the reduction or conversion of1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-oneto the product(R)-1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-olusing the listed engineered ketoreductase polypeptides compared to theactivity measured using ADH-LK (SEQ ID NO:2).

TABLE 1 Activity Nucleic Acid Polypeptide Mutations from ADH-LK RankingSEQ ID NO. SEQ ID NO. (SEQ ID NO: 2) (see legend)  5 6 L17Q; A94F; S96Y;F147L; L153G; L199I +++  7 8 G53D; S96G +  9 10 S96G + 11 12 S96G;K109R + 13 14 S96Y + 15 16 A94F; S96P + 17 18 S96F + 19 20 T54A; A94F;S96G; K109R; F147L; L153A; +++ D233G 21 22 V43A; V60A; A94G; F147L +++23 24 V43A; F74L; S96I; F147L; L153G; L199K; +++ I223V 25 26 V43A; F74L;S96G; F147L; L153A; D197G; ++ L199I; A202L 27 28 V43A; F74L; A94G; S96G;F147L; L153A; +++ D197G; L199I; A202L; I223V 29 30 V43A; F74L; A94G;S96Y; F147L; L153A; ++ K211R; I223V 31 32 V43A; F74L; A94G; F147L;L199K; A202I ++ 33 34 V43A; F74L; A94G; F147L; L199I; A202I; ++ M206F;I223V 35 36 V43A; F74L; A94F; S96Y; L153A; I165T ++ 37 38 V43A; A94G;F147L +++ 39 40 V43A; A94G; E106G; F147L; A202L ++ 41 42 V43A; A94F;V95I; S96G; F147L; L153G; +++ D197G; D233G 43 44 V43A; A94F; S96D;F147L; L153G; D197G; +++ D233G 45 46 V43A; A94F; S96G; F147L; L153G;D197G; +++ L199Q; D233G 47 48 V43A; A94F; S96G; F147L; L153G; D197G; +++L199R; D233G 49 50 V43A; A94F; S96G; F147L; L153G; D197G; ++ D233G 51 52V43A; A94F; S96G; K97G; F147L; L153G; +++ D197G; M205T; D233G 53 54V43A; A94F; S96Y; L153G; D197G; L199N; ++ A202I; I223V 55 56 V43A; T54A;A94G; S96G; F147L; I223V +++ 57 58 F74L; A94G; S96G; F147L; L153A;A202I; ++ M206F 59 60 F74L; A94G; S96G; F147L; L153A; D197G; ++ L199K 6162 F74L; A94G; F147L; L199K; A202I +++ 63 64 F74L; A94G; F147L; D197G;I223V ++ 65 66 A94G; F147L; L199I; A202I ++ 67 68 A94G; F147L; L199I;A202L; M206F; K211R ++ 69 70 A94F; V95I; S96Y; F147L; L153G +++   7′ 72A94F; V95L; S96Y; F147L; L153G +++ 73 74 A94F; S96G; F147L; L153A;L199V; D233G +++ 75 76 A94F; S96Y; L124Q; F147L; L153G +++ 77 78 A94F;S96Y; F147L; L153G ++ 79 80 A94F; S96Y; K109R; F147L; L153A; D233G +++81 82 A94F; S96F; K109R; F147L; L153A; D233G +++ 83 84 T77A; A94F; S96Y;F147L; L153G; L199W +++ 85 86 T54A; A94F; S96G; F147L; L153A; D233G +++87 88 T54A; A94F; S96G; F147L; L153A; L199V; +++ D233G 89 90 T54A; A94F;S96Y; F147L; L153A; D233G +++ 91 92 T54A; A94F; S96Y; F147L; L153G;D233G +++ 93 94 T54A; A94F; S96Y; K109R; F147L; L153A; +++ L199I; D233G;95 96 T54A; A94F; S96Y; K109R; F147L; L153A; +++ L199R 97 98 T54A; A94F;S96Y; K109R; F147L; L153A; +++ D233G 99 100 T54A; A94F; K109R; F147L;L153A; D233G +++ 101  102 T54A; A94F; K109R; F147L; L153A; L199K; +++N221D; D233G 103  104 T54A; A94F; S96F; K109R; F147L; L153A; +++ D233G105  106 K49R; A94G; F147L; D197G; A202G ++ 107  108 V43A; F74L; A94F;S96Y; L153G ++ 109  110 T54A; A94F; S96G; K109R; F147L; L153A; +++L199K; D233G 111  112 I11V; A94F + 113  114 A94G + ¹Activity Ranking: +Up to 20-fold improved in activity compared to ADH-LK. ++ From 21- to80-fold improved in activity compared to ADH-LK. +++ More than 80-foldimproved in activity compared to ADH-LK.

In some embodiments, the engineered ketoreductase enzymes are selectedfrom the amino acid sequences recited in Table 2. Table 2 ranks theenzyme activity measured for the reduction or conversion of acetophenoneto (R)-1-phenylethanol using the listed engineered ketoreductasepolypeptides derived from ADH-LK compared to the activity measured usingADH-LK (SEQ ID NO:2).

TABLE 2 Mutations from ADH-LK Activity Nucleic Acid Polypeptide (SEQ IDNO: 2) or ADH- Ranking¹ SEQ ID NO. SEQ ID NO. LB (SEQ ID NO: 4) (seelegend) 115 116 ADH-LB: A96G 117 118 ADH-LB: N96F 119 120 ADH-LK: S96F+++ 121 122 ADH-LK: S96G; F147L; +++ L199N 123 124 ADH-LK: S96F; F147L+++ 125 126 ADH-LK: S96Y; F147L +++ 127 128 ADH-LK: S96I +++ 129 130ADH-LK: A94H ++ 131 132 ADH-LK: S96C ++ 133 134 ADH-LK: S96W ++ 135 136ADH-LK: S96I; F147L +++ 137 138 ADH-LK: A94S; F147L +++ ¹ActivityRanking: Acetophenone conversion to (R) 1-phenylethanol in the method ofExample 16: + <70% conversion; ++ 70-90% conversion; +++ >90%conversion.

In some embodiments, the ketoreductase polypeptide comprises an aminoacid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2 orSEQ ID NO:4, wherein the amino acid sequence comprises any one of theset of mutations contained in any one of the polypeptide sequenceslisted in Table 1 or Table 2. In some embodiments, the ketoreductasepolypeptide comprises any one of the set of the amino acid residues atthe specified residue positions of Table I or Table 2 and which differsfrom the corresponding sequence from about 1-2, 1-3, 1-4, 1-5, 1-6, 1-7,1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-16, 1-18, 1-20, or 1-25 amino acidpositions. In some embodiments, the ketoreductase polypeptide comprisesany one of the set of mutations listed in Table 1 or Table 2, andadditionally from about 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10,1-11, 1-12, 1-14, 1-16, 1-18, 1-20, or 1-25 conservative substitutionsat other residues. Thus, in some embodiments, the ketoreductasepolypeptide can differ in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16,18, 20, or 25 amino acid positions as compared to the amino acidsequences in Table 1 or Table 2.

In some embodiments, the ketoreductase polypeptide can comprise a regionhaving an amino acid sequence that is at least about 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to aregion or domain thereof, such as residues 90-233, of SEQ ID NO: 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, or 118. In some embodiments, the ketoreductase polypeptidescan comprise a region having an amino acid sequence that is at leastabout 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a region or domain thereof, such as residues90-233 of SEQ ID NO: 120, 122, 124, 126, 128, 130, 132, 134, 136, or138.

In some embodiments, the ketoreductase polypeptide is selected from SEQID NOS: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,108, 110, 112, 114, 116, and 118. In some embodiments, the ketoreductasepolypeptide is selected from SEQ ID NOS: 120, 122, 124, 126, 128, 130,132, 134, 136, and 138.

In some embodiments, where the ketoreductase polypeptide comprises anamino acid sequence with an aromatic acid or G at the residuecorresponding to residue 94 of SEQ ID NO:2 or SEQ ID NO:4, theketoreductase polypeptide can be selected from the amino acid sequencesof SEQ ID NOS: 6, 16, 20, 22, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, and 128.

In some embodiments, where the ketoreductase polypeptide comprises anamino acid sequence with an amino acid other than S/N at the residuecorresponding to residue 96 of SEQ ID NO:2 or SEQ ID NO:4, theketoreductase polypeptide can be selected from the amino acid sequencesof SEQ ID NOS: 6, 8, 10, 12, 14, 16, 18, 20, 24, 26, 28, 30, 36, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 104, 108, 110, 118, 120, 122, 124, 126, 128, 132,134, and 136.

In some embodiments, segments of the ketoreductase polypeptides can bedeleted to generate polypeptide fragments. The term “fragment” as usedherein refers to a polypeptide that has an amino-terminal and/orcarboxy-terminal deletion, but where the remaining amino acid sequenceis identical to the corresponding positions in the sequence. Fragmentscan be at least 14 amino acids long, at least 20 amino acids long, atleast 50 amino acids long or longer. In some embodiments, the fragmentsare up to 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% of the full-length recombinant ketoreductasepolypeptide above, including fragments of the ketoreductase polypeptidesof SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, and 118. In some embodiments, thefragments are up to 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full-length recombinantketoreductase polypeptide above, including fragments of theketoreductase polypeptides of SEQ ID NO: 120, 122, 124, 126, 128, 130,132, 134, 136, and 138.

The improved ketoreductase enzymes may be present within a cell, presentin the cellular medium, or prepared in various forms, such as lysates orisolated preparations. As such, in some embodiments, the improvedketoreductase enzyme can be an isolated polypeptide. The term “isolatedpolypeptide” refers to a polypeptide which is substantially separatedfrom other contaminants that naturally accompany it, e.g., protein,lipids, and polynucleotides. The term embraces polypeptides which havebeen removed or purified from their naturally-occurring environment orexpression system (e.g., host cell or in vitro synthesis).

In some embodiments, the isolated improved ketoreductases polypeptide isa substantially pure polypeptide composition. The term “substantiallypure polypeptide” refers to a composition in which the polypeptidespecies is the predominant species present (i.e., on a molar or weightbasis it is more abundant than any other individual macromolecularspecies in the composition), and is generally a substantially purifiedcomposition when the object species comprises at least about 50 percentof the macromolecular species present by mole or % weight. Generally, asubstantially pure ketoreductase composition will comprise about 60% ormore, about 70% or more, about 80% or more, about 90% or more, about 95%or more, and about 98% or more of all macromolecular species by mole or% weight present in the composition. In some embodiments, the objectspecies is purified to essential homogeneity (i.e., contaminant speciescannot be detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies. Solvent species, small molecules (<500 Daltons), and elementalion species are not considered macromolecular species.

6.3 POLYNUCLEOTIDES ENCODING ENGINEERED KETOREDUCTASES

In another aspect, the present disclosure provides polynucleotidesencoding the engineered ketoreductase polypeptides. The polynucleotidesmay be operatively linked to one or more heterologous regulatory orcontrol sequences that control gene expression to create a recombinantpolynucleotide capable of expressing the polypeptide. Expressionconstructs containing a heterologous polynucleotide encoding theengineered ketoreductase can be introduced into appropriate host cellsto express the corresponding ketoreductase.

Because of the knowledge of the codons corresponding to the variousamino acids, availability of a polypeptide sequence provides adescription of all the polynucleotides capable of encoding the subjectpolypeptide. The degeneracy of the genetic code, where the same aminoacids are encoded by alternative or synonymous codons allows anextremely large number of nucleic acids to be made, all of which encodethe improved ketoreductase enzymes disclosed herein. Thus, havingidentified a particular amino acid sequence, those skilled in the artcould make any number of different nucleic acids by simply modifying thesequence of one or more codons in a way which does not change the aminoacid sequence of the protein. In this regard, the present disclosurespecifically contemplates each and every possible variation ofpolynucleotides that could be made by selecting combinations based onthe possible codon choices, and all such variations are to be consideredspecifically disclosed for any polypeptide disclosed herein, includingthe amino acid sequences presented in Table 1 and Table 2. As such, thepolynucleotides of the present disclosure include any and all possiblepolynucleotide sequences that encode the ketoreductase polypeptide ofSEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,136, or 138.

In some embodiments, the polynucleotides encoding the ketoreductasesenzymes may be codon optimized for optimal production from the hostorganism selected for expression. For example, preferred codons used inbacteria are used to express the gene in bacteria; preferred codons usedin yeast are used for expression in yeast; and preferred codons used inmammals are used for expression in mammalian cells. By way of example,the polynucleotide of SEQ ID NO: 1 has been codon optimized forexpression in E. coli, but otherwise encodes the naturally occurringketoreductase of Lactobacillus kefir.

In some embodiments, all codons need not be replaced to optimize thecodon usage of the ketoreductases since the natural sequence willcomprise preferred codons and because use of preferred codons may not berequired for all amino acid residues. Consequently, codon optimizedpolynucleotides encoding the ketoreductase enzymes may contain preferredcodons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of codonpositions of the full length coding region.

In some embodiments, the polynucleotides encoding the engineeredketoreductases are selected from SEQ ID NOS: 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, and 117. Insome embodiments, the polynucleotides encoding the engineeredketoreductases are selected from SEQ ID NOS: 119, 121, 123, 125, 127,129, 131, 133, 135, and 137.

These polynucleotides encode the corresponding polypeptides representedby the amino acid sequences listed in Table 1 and Table 2, which werederived by subjecting the E. coli codon optimized Lactobacillus kefirgene to directed gene evolution techniques described herein.

In some embodiments, the polynucleotides comprise polynucleotides thatencode the polypeptides described herein but have about 80% or moresequence identity, about 85% or more sequence identity, about 90% ormore sequence identity, about 91% or more sequence identity, about 92%or more sequence identity, about 93% or more sequence identity, about94% or more sequence identity, about 95% or more sequence identity,about 96% or more sequence identity, about 97% or more sequenceidentity, about 98% or more sequence identity, or about 99% or moresequence identity at the nucleotide level to a reference polynucleotideencoding an engineered ketoreductase. In some embodiments, the referencepolynucleotide is selected from polynucleotide sequences of SEQ ID NOS:5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, and 117. In some embodiments, the referencepolynucleotide is selected from sequences of SEQ ID NOS: 119, 121, 123,125, 127, 129, 131, 133, 135, and 137.

In some embodiments, the polynucleotide encodes the polypeptidedisclosed herein and hybridizes under defined conditions, such asmoderately stringent or highly stringent conditions, to the complementof a sequence encoding an engineered ketoreductase enzyme of the presentdisclosure. As such, in some embodiments, the polynucleotides encodingthe ketoreductase polypeptides comprises a polynucleotide thathybridizes under highly stringent conditions to a polynucleotideselected from SEQ ID NOS: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, and 117. In some embodiments,the polynucleotides encoding the ketoreductase polypeptides comprises apolynucleotide that hybridizes under highly stringent conditions to apolynucleotide selected from SEQ ID NOS: 119, 121, 123, 125, 127, 129,131, 133, 135, and 137.

An isolated polynucleotide encoding an improved ketoreductasepolypeptide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the isolatedpolynucleotide prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotides and nucleic acid sequences utilizingrecombinant DNA methods are well known in the art. Guidance is providedin Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3^(rd)Ed., Cold Spring Harbor Laboratory Press; and Current Protocols inMolecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998,updates to 2006.

In some embodiments, the control sequence may be an appropriate promotersequence, which can be obtained from genes encoding extracellular orintracellular polypeptides either homologous or heterologous to the hostcell. For bacterial host cells, suitable promoters for directingtranscription of the nucleic acid constructs of the present disclosure,include the promoters obtained from the E. coli lac operon, Streptomycescoelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene(sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillusamyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformispenicillinase gene (penP), Bacillus subtilis xylA and xylB genes, andprokaryotic beta-lactamase gene (VIIIa-Kamaroff et al., 1978, Proc.Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoeret al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Further promotersare described in “Useful proteins from recombinant bacteria” inScientific American, 1980, 242:74-94; and in Sambrook et al., supra.

For filamentous fungal host cells, suitable promoters for directing thetranscription of the nucleic acid constructs of the present disclosureinclude promoters obtained from the genes for Aspergillus oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters can be from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8:423-488.

In some embodiments, the control sequence may also be a suitabletranscription terminator sequence, a sequence recognized by a host cellto terminate transcription. The terminator sequence is operably linkedto the 3′ terminus of the nucleic acid sequence encoding thepolypeptide. Any terminator which is functional in the host cell ofchoice may be used in the present invention.

For example, exemplary transcription terminators for filamentous fungalhost cells can be obtained from the genes for Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease.

Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C(CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

In some embodiments, the control sequence may also be a suitable leadersequence, a nontranslated region of an mRNA that is important fortranslation by the host cell. The leader sequence is operably linked tothe 5′ terminus of the nucleic acid sequence encoding the polypeptide.Any leader sequence that is functional in the host cell of choice may beused. Exemplary leaders for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase and Aspergillusnidulans triose phosphate isomerase. Suitable leaders for yeast hostcells are obtained from the genes for Saccharomyces cerevisiae enolase(ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase,Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase(ADH2/GAP).

In some embodiments, the control sequence may also be a polyadenylationsequence, a sequence operably linked to the 3′ terminus of the nucleicacid sequence and which, when transcribed, is recognized by the hostcell as a signal to add polyadenosine residues to transcribed mRNA. Anypolyadenylation sequence which is functional in the host cell of choicemay be used in the present invention. Exemplary polyadenylationsequences for filamentous fungal host cells can be from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,Aspergillus nidulans anthranilate synthase, Fusarium oxysporumtrypsin-like protease, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are described by Guo andSherman, 1995, Mol Cell Bio 15:5983-5990.

In some embodiments, the control sequence may also be a signal peptidecoding region that codes for an amino acid sequence linked to the aminoterminus of a polypeptide and directs the encoded polypeptide into thecell's secretory pathway. The 5′ end of the coding sequence of thenucleic acid sequence may inherently contain a signal peptide codingregion naturally linked in translation reading frame with the segment ofthe coding region that encodes the secreted polypeptide. Alternatively,the 5′ end of the coding sequence may contain a signal peptide codingregion that is foreign to the coding sequence. The foreign signalpeptide coding region may be required where the coding sequence does notnaturally contain a signal peptide coding region.

Alternatively, the foreign signal peptide coding region may simplyreplace the natural signal peptide coding region in order to enhancesecretion of the polypeptide. However, any signal peptide coding regionwhich directs the expressed polypeptide into the secretory pathway of ahost cell of choice may be used in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NClB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiol Rev 57: 109-137.

Effective signal peptide coding regions for filamentous fungal hostcells can be the signal peptide coding regions obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells can be from the genes forSaccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

In some embodiments, the control sequence may also be a propeptidecoding region that codes for an amino acid sequence positioned at theamino terminus of a polypeptide. The resultant polypeptide is known as aproenzyme or propolypeptide (or a zymogen in some cases). Apropolypeptide is generally inactive and can be converted to a matureactive polypeptide by catalytic or autocatalytic cleavage of thepropeptide from the propolypeptide. The propeptide coding region may beobtained from the genes for Bacillus subtilis alkaline protease (aprE),Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiaealpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthorathermophile lactase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude the lac, tac, and tip operator systems. In yeast host cells,suitable regulatory systems include, as examples, the ADH2 system orGAL1 system. In filamentous fungi, suitable regulatory sequences includethe TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter.

Other examples of regulatory or control sequences are those which allowfor gene amplification. In eukaryotic systems, these include thedihydrofolate reductase gene, which is amplified in the presence ofmethotrexate, and the metallothionein genes, which are amplified withheavy metals. In these cases, the nucleic acid sequence encoding theKRED polypeptide of the present invention would be operably linked withthe control sequence.

Thus, in another aspect, the present disclosure is also directed to arecombinant expression vector comprising a polynucleotide encoding anengineered ketoreductase polypeptide or a variant thereof, and one ormore expression regulating regions such as a promoter and a terminator,a replication origin, etc., depending on the type of hosts into whichthey are to be introduced. The various nucleic acid and controlsequences described above may be joined together to produce arecombinant expression vector which may include one or more convenientrestriction sites to allow for insertion or substitution of the nucleicacid sequence encoding the polypeptide at such sites. Alternatively, thenucleic acid sequence of the present disclosure may be expressed byinserting the nucleic acid sequence or a nucleic acid constructcomprising the sequence into an appropriate vector for expression. Increating the expression vector, the coding sequence is located in thevector so that the coding sequence is operably linked with theappropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

The expression vector may be an autonomously replicating vector, i.e., avector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The expression vector of the present invention preferably contains oneor more selectable markers, which permit easy selection of transformedcells. A selectable marker is a gene the product of which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markers arethe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers, which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol (Example 1) or tetracycline resistance.Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3,TRP1, and URA3.

Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Embodiments for use in an Aspergillus cell include the amdS and pyrGgenes of Aspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The expression vectors of the present invention preferably contain anelement(s) that permits integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome. For integration into the host cell genome, the vector mayrely on the nucleic acid sequence encoding the polypeptide or any otherelement of the vector for integration of the vector into the genome byhomologous or nonhomologous recombination.

Alternatively, the expression vector may contain additional nucleic acidsequences for directing integration by homologous recombination into thegenome of the host cell. The additional nucleic acid sequences enablethe vector to be integrated into the host cell genome at a preciselocation(s) in the chromosome(s). To increase the likelihood ofintegration at a precise location, the integrational elements shouldpreferably contain a sufficient number of nucleic acids, such as 100 to10,000 base pairs, preferably 400 to 10,000 base pairs, and mostpreferably 800 to 10,000 base pairs, which are highly homologous withthe corresponding target sequence to enhance the probability ofhomologous recombination. The integrational elements may be any sequencethat is homologous with the target sequence in the genome of the hostcell. Furthermore, the integrational elements may be non-encoding orencoding nucleic acid sequences. On the other hand, the vector may beintegrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are P15Aon (as shown in the plasmid of FIG. 5) or the origins of replication ofplasmids pBR322, pUC19, pACYC177 (which plasmid has the P15A ori), orpACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060,or pAM.beta.1 permitting replication in Bacillus. Examples of origins ofreplication for use in a yeast host cell are the 2 micron origin ofreplication, ARS1, ARS4, the combination of ARS1 and CEN3, and thecombination of ARS4 and CEN6. The origin of replication may be onehaving a mutation which makes it's functioning temperature-sensitive inthe host cell (see, e.g., Ehrlich, 1978, Proc Natl Acad. Sci. USA75:1433).

More than one copy of a nucleic acid sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleic acid sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleic acid sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleic acid sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

Many of the expression vectors for use in the present invention arecommercially available. Suitable commercial expression vectors includep3xFLAGTM™ expression vectors from Sigma-Aldrich Chemicals, St. LouisMo., which includes a CMV promoter and hGH polyadenylation site forexpression in mammalian host cells and a pBR322 origin of replicationand ampicillin resistance markers for amplification in E. coli. Othersuitable expression vectors are pBluescriptll SK(−)® and pBK-CMV, whichare commercially available from Stratagene, LaJolla Calif., and plasmidswhich are derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4(Invitrogen) or pPoly (Lathe et al., 1987, Gene 57:193-201).

6.4 HOST CELLS FOR EXPRESSION OF KETOREDUCTASE POLYPEPTIDES

In another aspect, the present disclosure provides a host cellcomprising a polynucleotide encoding an improved ketoreductasepolypeptide of the present disclosure, the polynucleotide beingoperatively linked to one or more control sequences for expression ofthe ketoreductase enzyme in the host cell. Host cells for use inexpressing the ketoreductase polypeptides encoded by the expressionvectors of the present invention are well known in the art and includebut are not limited to, bacterial cells, such as E. coli, Lactobacilluskefir, Lactobacillus brevis, Streptomyces and Salmonella typhimuriumcells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiaeor Pichia pastoris (ATCC Accession No. 201178)); insect cells such asDrosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS,BHK, 293, and Bowes melanoma cells; and plant cells. Appropriate culturemediums and growth conditions for the above-described host cells arewell known in the art.

Polynucleotides for expression of the ketoreductase may be introducedinto cells by various methods known in the art. Techniques include amongothers, electroporation, biolistic particle bombardment, liposomemediated transfection, calcium chloride transfection, and protoplastfusion. Various methods for introducing polynucleotides into cells willbe apparent to the skilled artisan.

An exemplary host cell is Escherichia coli W3110. The expression vectorwas created by operatively linking a polynucleotide encoding an improvedketoreductase into the plasmid pCK110900 operatively linked to the lacpromoter under control of the lacI repressor. The expression vector alsocontained the P15a origin of replication and the chloramphenicolresistance gene. Cells containing the subject polynucleotide inEscherichia coli W3110 were isolated by subjecting the cells tochloramphenicol selection.

6.5 METHODS OF GENERATING ENGINEERED KETOREDUCTASE POLYPEPTIDES

To make the improved ketoreductase polynucleotides and polypeptides ofthe present disclosure, the naturally-occurring ketoreductase enzymethat catalyzes the reduction reaction is obtained from Lactobacilluskefir or Lactobacillus brevis. In some embodiments, the parentpolynucleotide sequence is codon optimized to enhance expression of theketoreductase in a specified host cell. As an illustration, the parentalpolynucleotide sequence encoding the wild-type ketoreductase polypeptideof Lactobacillus kefir was constructed from oligonucleotides preparedbased upon the known polypeptide sequence of Lactobacillus kefir KREDsequence available in Genbank database (Genbank accession no. AAP94029GI:33112056). The parental polynucleotide sequence, designated as SEQ IDNO: 1, was codon optimized for expression in E. coli and thecodon-optimized polynucleotide cloned into an expression vector, placingthe expression of the ketoreductase gene under the control of the lacpromoter and lad repressor gene. Clones expressing the activeketoreductase in E. coli were identified and the genes sequenced toconfirm their identity. The sequence designated (SEQ ID NO: 1) was theparent sequence utilized as the starting point for all experiments andlibrary construction of engineered ketoreductases evolved from theLactobacillus kefir ketoreductase.

The engineered ketoreductases can be obtained by subjecting thepolynucleotide encoding the naturally occurring ketoreductase tomutagenesis and/or directed evolution methods, as discussed above. Anexemplary directed evolution technique is mutagenesis and/or DNAshuffling as described in Stemmer, 1994, Proc Natl Acad Sci USA91:10747-10751; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO00/42651; WO 01/75767 and U.S. Pat. No. 6,537,746. Other directedevolution procedures that can be used include, among others, staggeredextension process (StEP), in vitro recombination (Zhao et al., 1998,Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell et al., 1994, PCRMethods Appl. 3:S136-S140), and cassette mutagenesis (Black et al.,1996, Proc Natl Acad Sci USA 93:3525-3529).

The clones obtained following mutagenesis treatment are screened forengineered ketoreductases having a desired improved enzyme property.Measuring enzyme activity from the expression libraries can be performedusing the standard biochemistry technique of monitoring the rate ofdecrease (via a decrease in absorbance or fluorescence) of NADH or NADPHconcentration, as it is converted into NAD⁺ or NADP⁺. In this reaction,the NADH or NADPH is consumed (oxidized) by the ketoreductase as theketoreductase reduces a ketone substrate to the corresponding hydroxylgroup. The rate of decrease of NADH or NADPH concentration, as measuredby the decrease in absorbance or fluorescence, per unit time indicatesthe relative (enzymatic) activity of the ketoreductase polypeptide in afixed amount of the lysate (or a lyophilized powder made therefrom).Where the improved enzyme property desired is thermal stability, enzymeactivity may be measured after subjecting the enzyme preparations to adefined temperature and measuring the amount of enzyme activityremaining after heat treatments. Clones containing a polynucleotideencoding a ketoreductase are then isolated, sequenced to identify thenucleotide sequence changes (if any), and used to express the enzyme ina host cell.

Where the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical litigationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides of theinvention can be prepared by chemical synthesis using, e.g., theclassical phosphoramidite method described by Beaucage et al., 1981, TetLett 22:1859-69, or the method described by Matthes et al., 1984, EMBOJ. 3:801-05, e.g., as it is typically practiced in automated syntheticmethods. According to the phosphoramidite method, oligonucleotides aresynthesized, e.g., in an automatic DNA synthesizer, purified, annealed,ligated and cloned in appropriate vectors. In addition, essentially anynucleic acid can be obtained from any of a variety of commercialsources, such as The Midland Certified Reagent Company, Midland, Tex.,The Great American Gene Company, Ramona, Calif., ExpressGen Inc.Chicago, Ill., Operon Technologies Inc., Alameda, Calif., and manyothers.

Engineered ketoreductase enzymes expressed in a host cell can berecovered from the cells and or the culture medium using any one or moreof the well known techniques for protein purification, including, amongothers, lysozyme treatment, sonication, filtration, salting-out,ultra-centrifugation, and chromatography. Suitable solutions for lysingand the high efficiency extraction of proteins from bacteria, such as E.coli, are commercially available under the trade name CelLytic B™ fromSigma-Aldrich of St. Louis Mo.

Chromatographic techniques for isolation of the ketoreductasepolypeptide include, among others, reverse phase chromatography highperformance liquid chromatography, ion exchange chromatography, gelelectrophoresis, and affinity chromatography. Conditions for purifying aparticular enzyme will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc.,and will be apparent to those having skill in the art.

In some embodiments, affinity techniques may be used to isolate theimproved ketoreductase enzymes. For affinity chromatographypurification, any antibody which specifically binds the ketoreductasepolypeptide may be used. For the production of antibodies, various hostanimals, including but not limited to rabbits, mice, rats, etc., may beimmunized by injection with a compound. The compound may be attached toa suitable carrier, such as BSA, by means of a side chain functionalgroup or linkers attached to a side chain functional group. Variousadjuvants may be used to increase the immunological response, dependingon the host species, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (bacilli Calmette Guerin) andCorynebacterium parvum.

6.6 METHODS OF USING THE ENGINEERED KETOREDUCTASE ENZYMES AND COMPOUNDSPREPARED THEREWITH

The ketoreductase enzymes described herein can catalyze the reduction ofthe substrate1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-one,as represented by the structure of formula (I):

to the chiral alcohol product(R)-1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4,]triazine-6-yloxy]-propan-2-ol,as represented by structure of formula (II)

The above compounds useful in the synthesis of protein kinaseinhibitors, and are encompassed within a class of compound described inUS application No. US 2006/0004007, US 2006/0128709; US 2006/0257400;US20060264438; and US20060286646 (all publications incorporated hereinby reference). Thus, other compounds of the class with the requisiteketo group can be used as substrates for the ketoreductases disclosedherein.

In various embodiments, the methods can comprise contacting or mixingthe compound of formula (I) with a ketoreductase of the presentdisclosure under reaction conditions suitable for conversion of thesubstrate to the compound of structural formula (II). Exemplaryreactions conditions are described in the Examples. Other suitablereactions conditions will be apparent to the skilled artisan. Exemplaryketoreductase polypeptides that can be used in the method include, butare not limited to, SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, and 118.

In some embodiments, the ketoreductase polypeptides described herein arecapable of converting the substrate acetophenone, as represented bystructural formula (III):

to the corresponding chiral alcohol product, (R)-1-phenylethanol, asrepresented by structural formula (IV):

In various embodiments, the method can comprise contacting or mixing thecompound of formula (III) with a ketoreductase of the present disclosureunder reaction conditions suitable for conversion of the substrate tothe compound of structural formula (IV). Exemplary reactions conditionsare described in the Examples (see, e.g., Example 15). Other suitablereactions conditions will be apparent to the skilled artisan. Exemplaryketoreductase polypeptides that can be used in the method include, butare not limited to, SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, or 138. In some embodiments, the ketoreductasepolypeptides that can be used for reduction or conversion ofacetophenone to (R)1-phenylethanol is selected from the polypeptides ofSEQ ID NO: 120, 122, 124, 126, 128, 130, 132, 134, 136, or 138.

In some embodiments, the resultant product is enriched in a particularstereoisomer, i.e., compound (II) or compound (IV). As used herein, acompound is “enriched” in a particular stereoisomer when thatstereoisomer is present in excess over any other stereoisomer present inthe compound. A compound that is enriched in a particular stereoisomerwill typically comprise at least about 60% or more, 70% or more, 80% ormore, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more,99% or more, or 99.5% or more, of the specified stereoisomer. The amountof enrichment of a particular stereoisomer can be confirmed usingconventional analytical methods routinely used by those of skill in theart.

In some embodiments, the amount of undesired stereoisomers may be lessthan 10%, for example, less than 9%, less than 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, less than 3%, less than 2%, lessthan 1% or even less than 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.2%, or0.1%. Stereoisomerically enriched compounds that contain at least about99.5% or more of the desired stereoisomer are referred to herein as“substantially pure” stereoisomers. In some embodiments, compounds thatare substantially pure in a specified stereoisomer contain greater than99.0%, 99.2%, 99.4%, 99.6%, 99.8%, or even greater purity.Stereoisomerically enriched compounds that contain ≧99.9% of the desiredstereoisomer are referred to herein as “pure” stereoisomers.

As is known by those of skill in the art, ketoreductase-catalyzedreduction reactions typically require a cofactor. Reduction reactionscatalyzed by the engineered ketoreductase enzymes described herein alsotypically require a cofactor, although many embodiments of theengineered ketoreductases require far less cofactor than reactionscatalyzed with wild-type ketoreductase enzymes. As used herein, the term“cofactor” refers to a non-protein compound that operates in combinationwith a ketoreductase enzyme. Cofactors suitable for use with theengineered ketoreductase enzymes described herein include, but are notlimited to, NADP⁺ (nicotinamide adenine dinucleotide phosphate), NADPH(the reduced form of NADP⁺), NAD⁺ (nicotinamide adenine dinucleotide)and NADH (the reduced form of NAD⁺). Generally, the reduced form of thecofactor is added to the reaction mixture. The reduced NAD(P)H form canbe optionally regenerated from the oxidized NAD(P)⁺ form using acofactor regeneration system.

Suitable exemplary cofactor regeneration systems that may be employedinclude, but are not limited to, glucose and glucose dehydrogenase,formate and formate dehydrogenase, glucose-6-phosphate andglucose-6-phosphate dehydrogenase, a secondary (e.g., isopropanol)alcohol and secondary alcohol dehydrogenase, phosphite and phosphitedehydrogenase, molecular hydrogen and hydrogenase, and the like. Thesesystems may be used in combination with either NADP⁺/NADPH or NAD⁺/NADHas the cofactor. Electrochemical regeneration using hydrogenase may alsobe used as a cofactor regeneration system. See, e.g., U.S. Pat. Nos.5,538,867 and 6,495,023, both of which are incorporated herein byreference. Chemical cofactor regeneration systems comprising a metalcatalyst and a reducing agent (for example, molecular hydrogen orformate) are also suitable. See, e.g., PCT publication WO 2000/053731,which is incorporated herein by reference.

The terms “glucose dehydrogenase” and “GDH” are used interchangeablyherein to refer to an NAD⁺ or NADP⁺-dependent enzyme that catalyzes theconversion of D-glucose and NAD⁺ or NADP⁺ to gluconic acid and NADH orNADPH, respectively. Equation (1), below, describes the glucosedehydrogenase-catalyzed reduction of NAD⁺ or NADP⁺ by glucose.

Glucose dehydrogenases that are suitable for use in the practice of themethods described herein include both naturally occurring glucosedehydrogenases, as well as non-naturally occurring glucosedehydrogenases. Naturally occurring glucose dehydrogenase encoding geneshave been reported in the literature. For example, the Bacillus subtilis61297 GDH gene was expressed in E. coli and was reported to exhibit thesame physicochemical properties as the enzyme produced in its nativehost (Vasantha et al., 1983, Proc. Natl. Acad. Sci. USA 80:785). Thegene sequence of the B. subtilis GDH gene, which corresponds to GenbankAcc. No. M12276, was reported by Lampel et al., 1986, J. Bacteriol.166:238-243, and in corrected form by Yamane et al., 1996, Microbiology142:3047-3056 as Genbank Acc. No. D50453. Naturally occurring GDH genesalso include those that encode the GDH from B. cereus ATCC 14579(Nature, 2003, 423:87-91; Genbank Acc. No. AE017013) and B. megaterium(Eur. J. Biochem., 1988, 174:485-490, Genbank Acc. No. X12370; J.Ferment. Bioeng., 1990, 70:363-369, Genbank Acc. No. GI216270). Glucosedehydrogenases from Bacillus sp. are provided in PCT publication WO2005/018579 as SEQ ID NOS: 10 and 12 (encoded by polynucleotidesequences corresponding to SEQ ID NOS: 9 and 11, respectively, of thePCT publication), the disclosure of which is incorporated herein byreference.

Non-naturally occurring glucose dehydrogenases may be generated usingknown methods, such as, for example, mutagenesis, directed evolution,and the like. GDH enzymes having suitable activity, whether naturallyoccurring or non-naturally occurring, may be readily identified usingthe assay described in Example 4 of PCT publication WO 2005/018579, thedisclosure of which is incorporated herein by reference. Exemplarynon-naturally occurring glucose dehydrogenases are provided in PCTpublication WO 2005/018579 as SEQ ID NOS: 62, 64, 66, 68, 122, 124, and126. The polynucleotide sequences that encode them are provided in PCTpublication WO 2005/018579 as SEQ ID NOS: 61, 63, 65, 67, 121, 123, and125, respectively. All of these sequences are incorporated herein byreference. Additional non-naturally occurring glucose dehydrogenasesthat are suitable for use in the ketoreductase-catalyzed reductionreactions disclosed herein are provided in US application publicationNos. 2005/0095619 and 2005/0153417, the disclosures of which areincorporated herein by reference.

Glucose dehydrogenases employed in the ketoreductase-catalyzed reductionreactions described herein may exhibit an activity of at least about 10μmol/min/mg and sometimes at least about 10² μmol/min/mg or about 10³μmol/min/mg, up to about 10⁴ μmol/min/mg or higher in the assaydescribed in Example 4 of PCT publication WO 2005/018579.

The ketoreductase-catalyzed reduction reactions described herein aregenerally carried out in a solvent. Suitable solvents include water,organic solvents (e.g., ethyl acetate, butyl acetate, 1-octanol,heptane, octane, methyl t-butyl ether (MTBE), toluene, and the like),and ionic liquids (e.g., 1-ethyl 4-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium hexafluorophosphate, and the like). In someembodiments, aqueous solvents, including water and aqueous co-solventsystems, are used.

Exemplary aqueous co-solvent systems have water and one or more organicsolvent. In general, an organic solvent component of an aqueousco-solvent system is selected such that it does not completelyinactivate the ketoreductase enzyme. Appropriate co-solvent systems canbe readily identified by measuring the enzymatic activity of thespecified engineered ketoreductase enzyme with a defined substrate ofinterest in the candidate solvent system, utilizing an enzyme activityassay, such as those described herein.

The organic solvent component of an aqueous co-solvent system may bemiscible with the aqueous component, providing a single liquid phase, ormay be partly miscible or immiscible with the aqueous component,providing two liquid phases. Generally, when an aqueous co-solventsystem is employed, it is selected to be biphasic, with water dispersedin an organic solvent, or vice-versa. Generally, when an aqueousco-solvent system is utilized, it is desirable to select an organicsolvent that can be readily separated from the aqueous phase. Ingeneral, the ratio of water to organic solvent in the co-solvent systemis typically in the range of from about 90:10 to about 10:90 (v/v)organic solvent to water, and between 80:20 and 20:80 (v/v) organicsolvent to water. The co-solvent system may be pre-formed prior toaddition to the reaction mixture, or it may be formed in situ in thereaction vessel.

The aqueous solvent (water or aqueous co-solvent system) may bepH-buffered or unbuffered. Generally, the reduction can be carried outat a pH of about 10 or below, usually in the range of from about 5 toabout 10. In some embodiments, the reduction is carried out at a pH ofabout 9 or below, usually in the range of from about 5 to about 9. Insome embodiments, the reduction is carried out at a pH of about 8 orbelow, often in the range of from about 5 to about 8, and usually in therange of from about 6 to about 8. The reduction may also be carried outat a pH of about 7.8 or below, or 7.5 or below. Alternatively, thereduction may be carried out a neutral pH, i.e., about 7.

During the course of the reduction reactions, the pH of the reactionmixture may change. The pH of the reaction mixture may be maintained ata desired pH or within a desired pH range by the addition of an acid ora base during the course of the reaction. Alternatively, the pH may becontrolled by using an aqueous solvent that comprises a buffer. Suitablebuffers to maintain desired pH ranges are known in the art and include,for example, phosphate buffer, triethanolamine buffer, and the like.Combinations of buffering and acid or base addition may also be used.

When the glucose/glucose dehydrogenase cofactor regeneration system isemployed, the co-production of gluconic acid (pKa=3.6), as representedin equation (3) causes the pH of the reaction mixture to drop if theresulting aqueous gluconic acid is not otherwise neutralized. The pH ofthe reaction mixture may be maintained at the desired level by standardbuffering techniques, wherein the buffer neutralizes the gluconic acidup to the buffering capacity provided, or by the addition of a baseconcurrent with the course of the conversion. Combinations of bufferingand base addition may also be used. Suitable buffers to maintain desiredpH ranges are described above. Suitable bases for neutralization ofgluconic acid are organic bases, for example amines, alkoxides and thelike, and inorganic bases, for example, hydroxide salts (e.g., NaOH),carbonate salts (e.g., NaHCO₃), bicarbonate salts (e.g., K₂CO₃), basicphosphate salts (e.g., K₂HPO₄, Na₃PO₄), and the like. The addition of abase concurrent with the course of the conversion may be done manuallywhile monitoring the reaction mixture pH or, more conveniently, by usingan automatic titrator as a pH stat. A combination of partial bufferingcapacity and base addition can also be used for process control.

When base addition is employed to neutralize gluconic acid releasedduring a ketoreductase-catalyzed reduction reaction, the progress of theconversion may be monitored by the amount of base added to maintain thepH. Typically, bases added to unbuffered or partially buffered reactionmixtures over the course of the reduction are added in aqueoussolutions.

In some embodiments, the co-factor regenerating system can comprises aformate dehydrogenase. The terms “formate dehydrogenase” and “FDH” areused interchangeably herein to refer to an NAD⁺ or NADP⁺-dependentenzyme that catalyzes the conversion of formate and NAD⁺ or NADP⁺ tocarbon dioxide and NADH or NADPH, respectively. Formate dehydrogenasesthat are suitable for use as cofactor regenerating systems in theketoreductase-catalyzed reduction reactions described herein includeboth naturally occurring formate dehydrogenases, as well asnon-naturally occurring formate dehydrogenases. Formate dehydrogenasesinclude those corresponding to SEQ ID NOS: 70 (Pseudomonas sp.) and 72(Candida boidinii) of PCT publication WO 2005/018579, which are encodedby polynucleotide sequences corresponding to SEQ ID NOS: 69 and 71,respectively, of PCT publication 2005/018579, the disclosures of whichare incorporated herein by reference. Formate dehydrogenases employed inthe methods described herein, whether naturally occurring ornon-naturally occurring, may exhibit an activity of at least about 1μmol/min/mg, sometimes at least about 10 μmol/min/mg, or at least about10² μmol/min/mg, up to about 10³ μmol/min/mg or higher, and can bereadily screened for activity in the assay described in Example 4 of PCTpublication WO 2005/018579.

As used herein, the term “formate” refers to formate anion (HCO₂),formic acid (HCO₂H), and mixtures thereof. Formate may be provided inthe form of a salt, typically an alkali or ammonium salt (for example,HCO₂Na, KHCO₂NH₄, and the like), in the form of formic acid, typicallyaqueous formic acid, or mixtures thereof. Formic acid is a moderateacid. In aqueous solutions within several pH units of its pKa (pKa=3.7in water) formate is present as both HCO₂ ⁻ and HCO₂H in equilibriumconcentrations. At pH values above about pH 4, formate is predominantlypresent as HCO₂ ⁻. When formate is provided as formic acid, the reactionmixture is typically buffered or made less acidic by adding a base toprovide the desired pH, typically of about pH 5 or above. Suitable basesfor neutralization of formic acid include, but are not limited to,organic bases, for example amines, alkoxides and the like, and inorganicbases, for example, hydroxide salts (e.g., NaOH), carbonate salts (e.g.,NaHCO₃), bicarbonate salts (e.g., K₂CO₃), basic phosphate salts (e.g.,K₂HPO₄, Na₃PO₄), and the like.

For pH values above about pH 5, at which formate is predominantlypresent as HCO₂ ⁻, Equation (2) below, describes the formatedehydrogenase-catalyzed reduction of NAD⁺ or NADP⁺ by formate.

When formate and formate dehydrogenase are employed as the cofactorregeneration system, the pH of the reaction mixture may be maintained atthe desired level by standard buffering techniques, wherein the bufferreleases protons up to the buffering capacity provided, or by theaddition of an acid concurrent with the course of the conversion.Suitable acids to add during the course of the reaction to maintain thepH include organic acids, for example carboxylic acids, sulfonic acids,phosphonic acids, and the like, mineral acids, for example hydrohalicacids (such as hydrochloric acid), sulfuric acid, phosphoric acid, andthe like, acidic salts, for example dihydrogenphosphate salts (e.g.,KH₂PO₄), bisulfate salts (e.g., NaHSO₄) and the like. Some embodimentsutilize formic acid, whereby both the formate concentration and the pHof the solution are maintained.

When acid addition is employed to maintain the pH during a reductionreaction using the formate/formate dehydrogenase cofactor regenerationsystem, the progress of the conversion may be monitored by the amount ofacid added to maintain the pH. Typically, acids added to unbuffered orpartially buffered reaction mixtures over the course of conversion areadded in aqueous solutions.

The terms “secondary alcohol dehydrogenase” and “sADH” are usedinterchangeably herein to refer to an NAD⁺ or NADP⁺-dependent enzymethat catalyzes the conversion of a secondary alcohol and NAD⁺ or NADP⁺to a ketone and NADH or NADPH, respectively. Equation (3), below,describes the reduction of NAD⁺ or NADP⁺ by a secondary alcohol,illustrated by isopropanol.

Secondary alcohol dehydrogenases that are suitable for use as cofactorregenerating systems in the ketoreductase-catalyzed reduction reactionsdescribed herein include both naturally occurring secondary alcoholdehydrogenases, as well as non-naturally occurring secondary alcoholdehydrogenases. Naturally occurring secondary alcohol dehydrogenasesinclude known alcohol dehydrogenases from, Thermoanerobium brockii,Rhodococcus etythropolis, Lactobacillus kefir, and Lactobacillus brevis,and non-naturally occurring secondary alcohol dehydrogenases includeengineered alcohol dehdyrogenases derived therefrom. Secondary alcoholdehydrogenases employed in the methods described herein, whethernaturally occurring or non-naturally occurring, may exhibit an activityof at least about 1 μmol/min/mg, sometimes at least about 10μmol/min/mg, or at least about 10² μmol/min/mg, up to about 10³μmol/min/mg or higher.

Suitable secondary alcohols include lower secondary alkanols andaryl-alkyl carbinols. Examples of lower secondary alcohols includeisopropanol, 2-butanol, 3-methyl-2-butanol, 2-pentanol, 3-pentanol,3,3-dimethyl-2-butanol, and the like. In one embodiment the secondaryalcohol is isopropanol. Suitable aryl-alkyl carbinols includeunsubstituted and substituted 1-arylethanols.

When a secondary alcohol and secondary alcohol dehydrogenase areemployed as the cofactor regeneration system, the resulting NAD⁺ orNADP⁺ is reduced by the coupled oxidation of the secondary alcohol tothe ketone by the secondary alcohol dehydrogenase. Some engineeredketoreductases also have activity to dehydrogenate a secondary alcoholreductant. In some embodiments using secondary alcohol as reductant, theengineered ketoreductase and the secondary alcohol dehydrogenase are thesame enzyme.

In carrying out embodiments of the ketoreductase-catalyzed reductionreactions described herein employing a cofactor regeneration system,either the oxidized or reduced form of the cofactor may be providedinitially. As described above, the cofactor regeneration system convertsoxidized cofactor to its reduced form, which is then utilized in thereduction of the ketoreductase substrate.

In some embodiments, cofactor regeneration systems are not used. Forreduction reactions carried out without the use of a cofactorregenerating systems, the cofactor is added to the reaction mixture inreduced form.

In some embodiments, when the process is carried out using whole cellsof the host organism, the whole cell may natively provide the cofactor.Alternatively or in combination, the cell may natively or recombinantlyprovide the glucose dehydrogenase.

In carrying out the enantioselective reduction reactions describedherein, the engineered ketoreductase enzyme, and any enzymes comprisingthe optional cofactor regeneration system, may be added to the reactionmixture in the form of the purified enzymes, whole cells transformedwith gene(s) encoding the enzymes, and/or cell extracts and/or lysatesof such cells. The gene(s) encoding the engineered ketoreductase enzymeand the optional cofactor regeneration enzymes can be transformed intohost cells separately or together into the same host cell. For example,in some embodiments one set of host cells can be transformed withgene(s) encoding the engineered ketoreductase enzyme and another set canbe transformed with gene(s) encoding the cofactor regeneration enzymes.Both sets of transformed cells can be utilized together in the reactionmixture in the form of whole cells, or in the form of lysates orextracts derived therefrom. In other embodiments, a host cell can betransformed with gene(s) encoding both the engineered ketoreductaseenzyme and the cofactor regeneration enzymes.

Whole cells transformed with gene(s) encoding the engineeredketoreductase enzyme and/or the optional cofactor regeneration enzymes,or cell extracts and/or lysates thereof, may be employed in a variety ofdifferent forms, including solid (e.g., lyophilized, spray-dried, andthe like) or semisolid (e.g., a crude paste).

The cell extracts or cell lysates may be partially purified byprecipitation (ammonium sulfate, polyethyleneimine, heat treatment orthe like, followed by a desalting procedure prior to lyophilization(e.g., ultrafiltration, dialysis, and the like). Any of the cellpreparations may be stabilized by crosslinking using known crosslinkingagents, such as, for example, glutaraldehyde or immobilization to asolid phase (e.g., Eupergit C, and the like).

The solid reactants (e.g., enzyme, salts, etc.) may be provided to thereaction in a variety of different forms, including powder (e.g.,lyophilized, spray dried, and the like), solution, emulsion, suspension,and the like. The reactants can be readily lyophilized or spray driedusing methods and equipment that are known to those having ordinaryskill in the art. For example, the protein solution can be frozen at−80° C. in small aliquots, then added to a prechilled lyophilizationchamber, followed by the application of a vacuum. After the removal ofwater from the samples, the temperature is typically raised to 4° C. fortwo hours before release of the vacuum and retrieval of the lyophilizedsamples.

The quantities of reactants used in the reduction reaction willgenerally vary depending on the quantities of product desired, andconcomitantly the amount of ketoreductase substrate employed. Thefollowing guidelines can be used to determine the amounts ofketoreductase, cofactor, and optional cofactor regeneration system touse. Generally, keto substrates can be employed at a concentration ofabout 20 to 300 grams/liter using from about 50 mg to about 5 g ofketoreductase and about 10 mg to about 150 mg of cofactor. Those havingordinary skill in the art will readily understand how to vary thesequantities to tailor them to the desired level of productivity and scaleof production. Appropriate quantities of optional cofactor regenerationsystem may be readily determined by routine experimentation based on theamount of cofactor and/or ketoreductase utilized. In general, thereductant (e.g., glucose, formate, isopropanol, etc.) is utilized atlevels above the equimolar level of ketoreductase substrate to achieveessentially complete or near complete conversion of the ketoreductasesubstrate.

The order of addition of reactants is not critical. The reactants may beadded together at the same time to a solvent (e.g., monophasic solvent,biphasic aqueous co-solvent system, and the like), or alternatively,some of the reactants may be added separately, and some together atdifferent time points. For example, the cofactor regeneration system,cofactor, ketoreductase, and ketoreductase substrate may be added firstto the solvent.

For improved mixing efficiency when an aqueous co-solvent system isused, the cofactor regeneration system, ketoreductase, and cofactor maybe added and mixed into the aqueous phase first. The organic phase maythen be added and mixed in, followed by addition of the ketoreductasesubstrate. Alternatively, the ketoreductase substrate may be premixed inthe organic phase, prior to addition to the aqueous phase

Suitable conditions for carrying out the ketoreductase-catalyzedreduction reactions described herein include a wide variety ofconditions which can be readily optimized by routine experimentationthat includes, but is not limited to, contacting the engineeredketoreductase enzyme and substrate at an experimental pH and temperatureand detecting product, for example, using the methods described in theExamples provided herein.

The ketoreductase catalyzed reduction is typically carried out at atemperature in the range of from about 15° C. to about 75° C. For someembodiments, the reaction is carried out at a temperature in the rangeof from about 20° C. to about 55° C. In still other embodiments, it iscarried out at a temperature in the range of from about 20° C. to about45° C. The reaction may also be carried out under ambient conditions.

The reduction reaction is generally allowed to proceed until essentiallycomplete, or near complete, reduction of substrate is obtained.Reduction of substrate to product can be monitored using known methodsby detecting substrate and/or product. Suitable methods include gaschromatography, HPLC, and the like. Conversion yields of the alcoholreduction product generated in the reaction mixture are generallygreater than about 50%, may also be greater than about 60%, may also begreater than about 70%, may also be greater than about 80%, may also begreater than 90%, and are often greater than about 97%.

7. EXAMPLES

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

In the following description, wherever glucose dehydrogenase (GDH) isused, it is GDH CDX901, obtainable from Julich Chiral Solutions,Jüllich, Germany.

7.1 Example 1 Wild-type Ketoreductase Gene Acquisition and Constructionof Expression Vectors

Ketoreductase (KRED) encoding genes were designed for expression in E.coli based on the reported amino acid sequence of the ketoreductase anda codon optimization algorithm as described in Example 1 of U.S.provisional application Ser. No. 60/848,950, incorporated herein byreference. Genes were synthesized using oligonucleotides composed of 42nucleotides and cloned into expression vector pCK110900 (depicted asFIG. 3 in United States Patent Application Publication 20060195947)under the control of a lac promoter. The expression vector alsocontained the P15a origin of replication and the chloramphenicolresistance gene. Resulting plasmids were transformed into E. coli W3110using standard methods. Codon optimized genes and the encodingpolypeptides adh-LB gene (Genbank Acc. No.: GI:28400789) and adh-LK gene(Genbank Acc. No.: AAP94029.1; GI:33112056) can be found as SEQ ID NO:1and 3. The activity of the wild-type ketoreductases was confirmed asdescribed in U.S. provisional application Ser. No. 60/848,950. Otherketoreductases used herein, including codon optimized genes and theencoding polypeptides for Ydl124wp (Genbank Acc. No.:NP 010159.1;GI:6320079), adh-LB gene (Genbank Acc. No.: 1NXQ A; GI:30749782), adh-REgene (Genbank Acc. No.: AAN73270.1; GI:34776951), Yprlp gene (GenbankAcc. No.: NP_(—)010656.1; GI:6320576), Gre2p gene (Genbank Acc. No.:NP_(—)014490.1; GI:6324421) are also disclosed in U.S. provisionalapplication Ser. No. 60/848,950, which is incorporated herein byreference.

Polynucleotides encoding engineered ketoreductases of the presentdisclosure were likewise cloned into vector pCK110900 for expression inE. coli W3110.

7.2 Example 2 LC/MS/MS Assay for Substrate Specificity and Conversion

A single microbial colony of E. coli containing a plasmid with theketoreductase gene of interest was inoculated into 50 ml Luria Bertanibroth containing 30 μg/ml chloramphenicol and 1% glucose. Cells weregrown overnight (at least 16 hrs) in an incubator at 30° C. with shakingat 250 rpm. The culture was diluted into 250 ml Terrific Broth (12 g/Lbacto-tryptone, 24 g/L yeast extract, 4 ml/L glycerol, 65 mM potassiumphosphate, pH 7.0, 1 mM MgSO₄, 30 μg/ml chloramphenicol) in 1 literflask) to an optical density at 600 nm (OD600) of 0.2 and allowed togrow at 30° C. Expression of the ketoreductase gene was induced with 1mM IPTG when the OD600 of the culture is 0.6 to 0.8 and incubatedovernight (at least 16 hrs). Cells were harvested by centrifugation(5000 rpm, 15 min, 4° C.) and the supernatant discarded. The cell pelletwas resuspended with an equal volume of cold (4° C.) 100 mMtriethanolamine (chloride) buffer, pH 7.0 (including 2 mM MgSO₄ in thecase of ADH-LK and ADH-LB and engineered ketoreductases derivedtherefrom), and harvested by centrifugation as above. The washed cellswere resuspended in two volumes of the cold triethanolamine (chloride)buffer and passed through a French Press twice at 12000 psi whilemaintained at 4° C. Cell debris was removed by centrifugation (9000 rpm,45 min., 4° C.). The clear lysate supernatant was collected and storedat −20° C. Lyophilization of frozen clear lysate provided a dry powderof crude ketoreductase enzyme.

The activity of the wild-type ketoreductases was confirmed as describedU.S. provisional application Ser. No. 60/848,950. To a solution of 1 mL100 mM (sodium) phosphate buffer, pH 7.5, were added 10 mg ketoreductasepowder, 50 mg NAD(P)H, 100 μL isopropanol and 10 mg4′-chloroacetophenone or unsubstituted acetophenone. The reactionmixture was stirred at room temperature for 16 hours, then extractedwith 1 mL MTBE. A sample of the MTBE phase was analyzed by chiral HPLCfor the conversion of the 4′-chloroacetophenone and the enantiomericcomposition of the product, 1-(4′-chlorophenyl)ethanol.

7.3 Example 3 Production of Ketoreductases; Fermentation Procedure

In an aerated agitated 15 L fermenter, 6.0 L of growth medium containing0.88 g/L ammonium sulfate, 0.98 g/L of sodium citrate; 12.5 g/L ofdipotassium hydrogen phosphate trihydrate, 6.25 g/L of potassiumdihydrogen phosphate, 6.2 g/L of Tastone-154 yeast extract, 0.083 g/Lferric ammonium citrate, and 8.3 ml/L of a trace element solutioncontaining 2 g/L of calcium chloride dihydrate, 2.2 g/L of zinc sulfateseptahydrate, 0.5 g/L manganese sulfate monohydrate, 1 g/L cuproussulfate heptahydrate, 0.1 g/L ammonium molybdate tetrahydrate and 0.02g/L sodium tetraborate decahydrate was brought to a temperature of 30°C. The fermenter was inoculated with a late exponential culture of E.coli W3110, containing a plasmid with the ketoreductase gene ofinterest, grown in a shake flask as described in Example 3 to a startingOD600 of 0.5 to 2.0. The fermenter was agitated at 500-1500 rpm and airwas supplied to the fermentation vessel at 1.0-15.0 L/min to maintaindissolved oxygen level of 30% saturation or greater. The pH of theculture was controlled at 7.0 by addition of 20% v/v ammonium hydroxide.Growth of the culture was maintained by the addition of a feed solutioncontaining 500 g/L cerelose, 12 g/L ammonium chloride and 10.4 g/Lmagnesium sulfate heptahydrate. After the culture reached an OD600 of50, the expression of ketoreductase was induced by the addition ofisopropyl-b-D-thiogalactoside (IPTG) to a final concentration of 1 mM.The culture was grown for another 14 hours. The culture was then chilledto 4° C. and maintained at 4° C. until harvested. Cells were harvestedby centrifugation at 5000 G for 40 minutes in a Sorval RC12BP centrifugeat 4° C. Harvested cells were used directly in the following downstreamrecovery process or were stored at 4° C. until such use.

The cell pellet was resuspended in 2 volumes of 100 mM triethanolamine(chloride) buffer, pH 6.8, at 4° C. to each volume of wet cell paste.The intracellular ketoreductase was released from the cells by passingthe suspension through a homogenizer fitted with a two-stagehomogenizing valve assembly using a pressure of 12000 psig. The cellhomogenate was cooled to 4° C. immediately after disruption. A solutionof 10% w/v polyethyleneimine, pH 7.2, was added to the lysate to a finalconcentration of 0.5% w/v and stirred for 30 minutes. The resultingsuspension was clarified by centrifugation at 5000G in a standardlaboratory centrifuge for 30 minutes. The clear supernatant was decantedand concentrated ten fold using a cellulose ultrafiltration membranewith a molecular weight cut off of 30 Kd. The final concentrate wasdispensed into shallow containers, frozen at −20° C. and lyophilized topowder. The ketoreductase powder was stored at −80° C.

7.4 Example 4 Analytical Methods to Determine Conversion andEnantiomeric Excess of Compound (II)

Reversed Phase HPLC assay for conversion. The following HPLC method wasused to analyze the reduction of the compound of formula (I) to thecompound of formula (II) in high throughput:

Instrument: Agilent 1100 series HP

Method name: 1549-ISO

Column type: Eclipse XDB C 18

Column size: 2.1×50 mm

Packing size: 3.5 μm C18 Zorbax XDB

Run time 3 minutes

Mobile phase 50% acetylnitrile 50% 0.25% acetic acid

Flow Rate: 0.6 ml/min, Temperature: Ambient

Detection: UV 250 nm

Elution time: alcohol: 0.9 minute

-   -   ketone: 1.2 minute.

Chiral HPLC for determination of enantiomeric excess. The following HPLCmethod was used to separate and analyze the (S) and (R) enantiomers ofthe compound of formula (II):

Instrument: Agilent 1100 series HP

Column: Chiralpak AD-H, 250×4.6 mm,

Solvent: Isocratic, 80% A (Heptane) and 20% B (Heptane-Isopropanol50:50)

Flow Rate: 1 ml/min, Temperature: Ambient

Detection: UV 220 nm

Run Time: 45 min

Retention times were as follows 29.1 min for (I), 35.4 min for R-(II),and 38.7 min for S-(II). The R- and S-alcohols showed baselineseparation with 1:1 area ratio. 17.5 Example 5

Evaluation of Wild-Type Ketoreductases for Activity to Reduce Compound(I) using Glucose and Glucose Dehydrogenase for Cofactor Regeneration

Reaction mixtures containing 30 mg/L KRED, 12 mg/ml1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-j][1,2,4]triazin-6-yloxy]propan-2-one(I) (obtainable according to scheme 1 and Example 1 in WO06130657,incorporated herein by reference), 1.5 mg/mL GDH CDX901 (Julich ChiralSolutions, Jülich, Germany), 1 mM NADP⁺, 66 mg/mL glucose, 200 mMNaH₂PO₄/Na₂HPO₄ (pH 7), 100 mM triethanolamine/chloride buffer, pH 7.0,1 mM MgSO₄ in 1 ml reaction volume were incubated with stirring at roomtemperature for 20 hr, then extracted with 3 mL ethyl acetate andanalyzed by the methods as described in Example 4 or according toExample 2 of WO06130657. The results are shown in Table 3.

TABLE 3 Activity of wild-type KREDs on Compound (I) Enzyme ActivityEnantioselectivity GREc + S YDLC + S and R YPrR + S ADH-LK + R RhoC +S + indicates that activity was observed.

The wild-type ketoreductase, ADH-LK, provides R-(II) with >99.9%enantioselectivity (>99.9% e.e.).

The Example illustrates the evaluation of wild-type ketoreductases foractivity and enantioselectivity when used in combination with a cofactorregeneration system (glucose and glucose dehydrogenase). Wild-typeketoreductase, ADH-LK, provides the desired (R)-enantiomer.

7.6 Example 6 Evaluation of Wild-Type Ketoreductases for Activity toReduce Compound (I) Using Isopropyl Alcohol for Cofactor Regeneration

Reaction mixtures containing 15 mg/ml KRED; 2 mg/ml (I) in DMF or in THF(added by 10×-dilution from a 20 mg/ml stock solution), 2 mg/mL NADP+,0.4-0.5 ml IPA, 0.4 ml 100 mM triethanolamine/chloride buffer, pH 7.0, 1mM MgSO₄ in 1 ml reaction volume were incubated with stirring at roomtemperature for 8 hr (when DMF was used) or 16 hr (when THF was used).Samples were analyzed by the methods of Examples 4 and 5.

Under these reaction conditions ADH-LK from L. kefiri provided 21%conversion in DMF and 61% conversion in THF, while ADH-LB from L. brevisprovided 70% conversion in DMF and 26% conversion in THF

The Example illustrates the evaluation of wild-type Lactobacillusketoreductases for activity and enantioselectivity when used incombination with IPA for cofactor regeneration. Wild-type ketoreductase,ADH-LK, provides the desired (R)-enantiomer in high e.e. in the presenceof 10% THF.

7.7 Example 7 High Throughput HPLC Assay for Ketoreductase Activity onCompound (I) Using Glucose/Glucose Dehydrogenase for Co-Factor Recycling

Library colonies were picked using a Q-bot® robotic colony picker(Genetix USA, Inc., Beaverton, Oreg.) into 96-well shallow wellmicrotiter plates containing 180 μL Luria Bertani broth (LB), 1% glucoseand 30 μg/mL chloramphenicol (CAM). Cells were grown overnight at 37° C.with shaking at 250 rpm. 10 μL of this culture was then transferred into96-deep well plates containing 390 μL Terrific broth (TB) and 30 μg/mLCAM. After incubation of deep-well plates at 30° C. with shaking at 250rpm for 2.5 to 3 hours (OD₆₀₀ 0.6-0.8), recombinant gene expression bythe cell cultures was induced by isopropyl thiogalactoside (IPTG) to afinal concentration of 1 mM. The plates were then incubated at 30° C.with shaking at 250 rpm for overnight.

Cells were pelleted via centrifugation, resuspended in 300 μL lysisbuffer and lysed by shaking at room temperature for at least 2 hours.The lysis buffer contained 100 mM triethanolamine (chloride) buffer, pH7.0-7.2, 1 mg/mL lysozyme and 750 μg/mL polymixin B sulfate.

Ketoreductase activity was measured by transferring measured quantitiesof the lysis mixtures into the wells of microtiter plates containing 205μL an assay mixture containing 1.7 mg/mL GDH CDX901, 0.7 mg/ml NADP+,66.7 mg/mL glucose, 200 mM NaH₂PO₄/Na₂HPO₄ (pH 7), 100 mMtriethanolamine/chloride buffer, pH 7.0, 1 mM MgSO₄. Reactions wereinitiated by addition of 25 μL 2 mg/ml (I) in DMF (final concentrationof (I) is 0.2 mg/ml) and incubated at 25° C. for 1 hr. 500 μLacetonitrile was added per well, mixed well after which 200 μL wastransferred to a Solvinert (Millipore, Mass.) filter plate. The filtratewas collected in a Nunc round bottom plate by centrifugation of thesolvenert filter plate at 200 rpm for 1 minute. The sample plate wassealed to prevent evaporation of the solvent and analyzed by HPLCaccording to Example 4.

7.8 Example 8 High Throughput HPLC Assay for Ketoreductase Activity onCompound (I) Using IPA for Co-Factor Recycling

Cell pellets were prepared according to Example 8, resuspended in 150 μL200 mM triethanolamine/chloride buffer, pH 7.0 with 1 mM MgSO₄. 150 μLisopropylalcohol was added to the resuspended cells and after sealingthe plates, the cells were lysed by shaking at room temperature on anorbital shaker for at least 120 minutes.

Ketoreductase activity was measured by transferring measured quantitiesof the lysis mixtures into the wells of a deep-well (2 ml) microtiterplates containing 175 μL an assay mixture consisting of 100 mMtriethanolamine/chloride buffer, pH 7.0, 25% isopropyl alcohol (IPA), 2%acetone, 1 mM MgSO₄ and 0.7 mg/ml NADP+, as well as 50 μL of 5 mg/ml (I)in 50% THF/50% IPA.

Reactions were initiated by addition of 25 μL lysate (diluted with equalvolume of IPA if necessary), heat sealed, and incubated in a shakingincubator at 25 to 50° C. for 18 hr. At the end of the reaction 500 μLethyl acetate was added to each well, the plate resealed and shakenvigorously for at least 5 minutes on an orbital shaker. Plates werecentrifuged for 20 to 30 sec. at 4000 rpm. 140 μL acetonitrile and 70 μLof the ethylacetate reaction mix were transferred to a Solvinert filterplate (Millipore, Mass.) and the filtrates were collected in a Nuncround bottom plate by centrifugation of the solvenert filter plate at200 rpm for 3 minutes. The sample plate was sealed to preventevaporation of the solvent and analyzed by HPLC according to Example 4.

7.9 Example 9 High Throughput Fluorescence Prescreen for KetoreductasesActive on Isopropanol

Cells were grown, harvested and lysed according to Example 8.

In 96-well black microtiter plates 20 μL of sample (diluted in 100 mMtriethanolamine/chloride buffer, pH 7.0, 1 mM MgSO₄ if necessary) wasadded to 180 μL of an assay mixture consisting of 100 mMtriethanolamine/chloride buffer, pH 7.0, 2% isopropyl alcohol (IPA), 1mM MgSO₄ and reaction progress measured by following the reduction influorescence of NADP upon conversion to NADPH at 445 nm after excitationat 330 nm in a Flexstation (Molecular Devices, USA).

7.10 Example 10 High Throughput Screen for Acetone Stable Ketoreductases

Cell pellets were prepared according to Example 8, resuspended in 150 μL200 mM triethanolamine/chloride buffer, pH 7.0 with 1 mM Mg50₄. 150 μLof a mix containing 76% IPA, 20% THF and 4% acetone was added to theresuspended cells and after sealing the plates, the cells were lysed byshaking at room temperature on an orbital shaker for 18 hrs.

Ketoreductase activity was measured by transferring measured quantitiesof the lysis mixtures into the wells of a deep-well (2 ml) microtiterplates containing 175 μL an assay mixture consisting of 80 mMtriethanolamine/chloride buffer, pH 7.0, 26.2 to 37.1% isopropyl alcohol(IPA), 1.8% acetone, 1 mM MgSO₄ and 0.7 mg/ml NADP+, as well as 50 μL of5 mg/ml (I) in 50% THF/50% IPA.

Reactions were initiated by addition of 25 μL lysate (diluted with equalvolume of IPA if necessary), heat sealed, and incubated in a shakingincubator at 25 to 50° C. for 18 hr. At the end of the reaction 500 μLethyl acetate was added to each well, the plate resealed and shakenvigorously for at least 5 minutes on an orbital shaker. Plates werecentrifuged for 20 to 30 sec. at 4000 rpm (3220×g). 140 μL acetonitrileand 70 μL of the ethylacetate reaction mix were transferred to aSolvinert filter plate (Millipore, Mass.) and the filtrates werecollected in a Nunc round bottom plate by centrifugation of thesolvenert filter plate at 200 rpm for 3 minutes. The sample plate wassealed to prevent evaporation of the solvent and analyzed by HPLCaccording to Example 4.

7.11 Example 11 Improved Activity of Engineered Ketoreductases Derivedfrom Wild-Type ADH-LK for the Reduction of Compound (I) to (R)-2 UsingIsopropyl Alcohol for Cofactor Regeneration

Reaction mixtures containing 15 mg/ml KRED; 2 mg/ml (I) in THF, 2 mg/mLNADP+, 0.4 ml IPA, 0.5 ml 100 mM triethanolamine/chloride buffer, pH7.0, 1 mM MgSO₄ in 1 ml reaction volume were incubated with stirring atroom temperature for 16 hr. Samples were analyzed by the methods ofExamples 4 and 5.

Under these reaction conditions ADH-LK gave 61% conversion, ADH-LB gave26% conversion in THF, a ADH-LK variant with SEQ ID NO:114 gave 100%conversion and a ADH-LK variant with SEQ ID NO:18 gave 93% conversion.

When tested under similar reaction conditions but with 3 mg/ml KRED and1 mg/ml (I) in THF, conversions with ADH-LK, SEQ ID NO:114 and SEQ IDNO:18 after a 2 hr reaction time were 11, 100 and 23% respectively.Introduction of the mutation A94G from SEQ ID NO:114 into ADH-LBprovided SEQ ID NO:116. Ketoreductases with SEQ ID NO:114 and SEQ IDNO:116 have similar activity in 10% DMF.

This Example illustrates, by comparisons of ketoreductase amounts,reaction times, and conversions, that engineered ketoreductases derivedfrom the wild-type ketoreductase ADH-LK provide improved activitycompared to ketoreductase ADH-LK.

7.12 Example 12 Improved Conversion of Compound (I) by EngineeredKetoreductases Derived from ADH-LK

The conversion by engineered ADH-LK polypeptides was determined byincubating 1 g/L of various KRED under conditions as described inExample 7.

Table 4 gives the SEQ ID NO. corresponding to the ketoreductase powder,the number of amino acid mutations from the wild-type ADH-LK and theconversion of Compound (I) to (R)-2 in the 20 minute reaction.

TABLE 4 Improved activity of engineered ADH-LK variants KetoreductasesSEQ ID Mutations from NO. ADH-LK Conversion SEQ ID NO: 2 — + SEQ ID NO:8 2 ++++ SEQ ID NO: 10 1 ++++ SEQ ID NO: 12 2 ++++ SEQ ID NO: 14 1 +++SEQ ID NO: 16 2 +++ SEQ ID NO: 18 1 ++++ SEQ ID NO: 112 2 ++++ SEQ IDNO: 114 1 ++++ +: <20% conversion; ++: 25-75% conversion; +++: >75%conversion.

7.13 Example 13 Improved Conversion and Tolerance to Acetone ofEngineered Ketoreductases Derived from ADH-LK

The conversion and tolerance to acetone of engineered ADH-LK variantswas determined by incubating 1 g/L of various KRED with 0.5 g/L (I) in amixture of 10% THF, 40% IPA, 50% 100 mM triethanolamine-chloride, 1 mMMgSO₄ pH 8.0, 0.7 mg/ml NADP′ in the absence or presence of 2% acetonefor 20 min at room temperature followed by determination of conversionaccording to Example 4.

Table 5 gives the SEQ ID NO. corresponding to the ketoreductase powder,the number of amino acid mutations from the wild-type ADH-LK and theconversion of (I) to (R)-2 in the 20 minute reaction.

TABLE 5 Tolerance of engineered ADH-LK variant towards acetone.Ketoreductases Mutations Conversion Conversion SEQ ID NO. from ADH-LKwithout acetone with acetone SEQ ID NO: 114 1 76%   12% SEQ ID NO: 50 798%   62% SEQ ID NO: 78 4 99.10%   96.0% SEQ ID NO: 110 8 98.6%   98.2%

7.14 Example 14 Preparation of R-Isomer of Compound (II)

To a 20 ml sample vial (21 mm OD) containing a magnetic stir bar (4×12mm) was added 3.5 mg of NADP⁺ (mono sodium salt from Oriental Yeast,Japan), 25 mg of KRED with SEQ ID NO:78 and 500 mg of (I). A mixture of1.5 ml 2-methylTHF (Aldrich, USA), 1.0 ml isopropyl alcohol, and 2.5 ml100 mM triethanolamine/chloride (pH 8), 1 mM MgSO₄, was added to thesolids and the resulting three-phase mixture was stirred and heated (oilbath) at 40° C. for 24 hr. A sample was taken from the stirred mixturefor analysis according to Example 4 or according to Example 2 ofWO06130657 at which point (I) was completely converted to R-(II)of >99.9% e.e.

7.15 Example 15 High Throughput Chiral GC Assay for AcetophenoneReduction Using IPA for Co-Factor Recycling Chiral GC Analysis:

Instrument: Astec Chiraldex B-DP column (30 m×0.25 mm)

Temperature: 110° C.

Inlet temperature: 250° C.

Split: 1:100 Pressure: 15 psi Helium Detector: FID, 250° C.

Retention time Ketone: 6.6 minutes

-   -   (R)-alcohol: 9.1 minutes    -   (S)-alcohol: 9.5 minutes

7.16 Example 16 Improved Activity of Engineered Ketoreductases Derivedfrom Wild-Type Kefir for the Reduction of Acetophenone to(R)-1-Phenylethanol Using Isopropyl Alcohol for Cofactor Regeneration

Cell lysates were prepared by picking colonies using a Q-bot® roboticcolony picker (Genetix USA, Inc., Beaverton, Oreg.) into 96-well shallowwell microtiter plates containing 180 μL Luria Bertani broth (LB), 1%glucose and 30 μg/mL chloramphenicol (CAM). Cells were grown overnightat 37° C. with shaking at 250 rpm. 10 μL of this culture was thentransferred into 96-deep well plates containing 390 μL, Terrific broth(TB) and 30 μg/mL CAM. After incubation of deep-well plates at 30° C.with shaking at 250 rpm for 2.5 to 3 hours (OD₆₀₀ 0.6-0.8), recombinantgene expression by the cell cultures was induced by isopropylthiogalactoside (IPTG) to a final concentration of 1 mM. The plates werethen incubated at 30° C. with shaking at 250 rpm for overnight.

Cells were pelleted via centrifugation, resuspended in 300 μL lysisbuffer and lysed by shaking at room temperature for at least 2 hours.The lysis buffer contained 100 mM triethanolamine (chloride) buffer, pH7.0-7.2, 1 mg/mL lysozyme and 750 μg/mL polymixin B sulfate. To 100 μLcell lysate was added in 50 μL 100 mM Triethanolamine-HCl buffer, pH7.0, containing 0.5 mM NADP sodium salt, 300 μL isopropanol, and 50 μL50 g/L acetophenone in tetrahydrofuran (THF) and after sealing theplates, the cells were incubated by shaking at room temperature at 850rpm on an orbital shaker for 4 hrs.

The product of the reaction (1-phenylethanol) was extracted by adding 1mL of ethyl acetate to each sample, and after sealing the microtiterplate, shaking at 850 rpm at room temperature for 10 minutes. Plateswere centrifuged for 2 minutes at 4,000 rpm in a plate centrifuge(3220×g) at 4° C. and 200 μL of the organic phase from each well wastransferred to a shallow well plate and the plates sealed prior tochiral GC analysis.

Table 6 gives the SEQ ID NO. corresponding to the ketoreductase, thenumber of amino acid mutations from the wild-type KEFc (ADH-LK) and theconversion of acetophenone to (R)-1-phenylethanol.

TABLE 6 Conversion of acetophenone. Number of amino Ketoreductases acidmutations from SEQ ID NO. ADH-LK Conversion ADH-LK — + SEQ ID NO: 16 2++ SEQ ID NO: 66 4 ++ SEQ ID NO: 120 1 +++ SEQ ID NO: 122 3 +++ SEQ IDNO: 124 2 +++ SEQ ID NO: 126 2 +++ SEQ ID NO: 128 1 +++ SEQ ID NO: 130 1++ SEQ ID NO: 132 1 ++ SEQ ID NO: 134 1 ++ SEQ ID NO: 136 2 +++ SEQ IDNO: 138 2 +++ +: <70% conversion; ++: 70-90% conversion; +++: >90%conversion.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. A recombinant ketoreductase polypeptide capableof converting the compound1-[4-(4-fluoro2-methyl-1H-indol-5-yloxy)-5-methyl-pyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-propan-2-oneto1-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-propan-2-ol,with at least 1.5 times the activity of the polypeptide of SEQ ID NO:2,the polypeptide comprising an amino acid sequence with at least 90%sequence identity to SEQ ID NO:2 or SEQ ID NO:4 and having (a) anaromatic amino acid or G at the amino acid residue corresponding toresidue 94 of SEQ ID NO:2 or SEQ ID NO:4, or (b) an amino acid otherthan S and N at the amino acid residue corresponding to residue 96 ofSEQ ID NO:2 or SEQ ID NO:4.
 2. The recombinant polypeptide of claim 1 inwhich the amino acid sequence comprises an aromatic amino acid or G atthe amino acid residue corresponding to residue 94 of SEQ ID NO:2 or SEQID NO:4.
 3. The recombinant polypeptide of claim 2 in which the residue94 is F, W, H, or Y.
 4. The recombinant polypeptide of claim 2 in whichthe amino acid sequence further comprises one or more features selectedfrom: residue 96 is any amino acid other than S/N; residue 153 is analiphatic amino acid residue other than L; residue 199 is any amino acidresidue other than L; residue 202 is G or an aliphatic amino acidresidue other than A; and residue 206 is an aromatic amino acid residue.5. The recombinant polypeptide of claim 4 in which the amino acidsequence has one or more of features selected from: residue 153 is G orA; residue 199 is K, I, N, R, V, Q, or W; residue 202 is I, L, or G; andresidue 206 is F.
 6. The recombinant polypeptide of any one of claims 1to 5 in which the amino acid sequence further comprises one or more ofthe features selected from: residue 49 is a polar amino acid residueother than K; residue 53 is an acidic amino acid residue; residue 54 isa small or aliphatic amino acid residue other than T/P; residue 60 is analiphatic amino acid residue other than V; residue 95 is an aliphaticamino acid other than V; residue 97 is a small amino acid or G; residue109 is a basic amino acid residue other than K; residue 147 is analiphatic amino acid residue; residue 165 is a hydroxyl or small aminoacid residue; residue 197 is a small amino acid residue or G; residue223 is an aliphatic amino acid residue other than L; and residue 233 isa small amino acid residue or G.
 7. The recombinant polypeptide of claim6 in which the amino acid sequence comprises one or more featuresselected from: residue 49 is R; residue 53 is D; residue 54 is A;residue 60 is A; residue 95 is L; residue 97 is G; residue 109 is R;residue 147 is L; residue 165 is T; residue 197 is G; residue 223 is V;and residue 233 is G.
 8. The recombinant polypeptide of claim 7 in whichthe amino acid sequence has one or more features selected from: residue54 is A; residue 109 is R; residue 147 is L; and residue 233 is G. 9.The recombinant polypeptide of claim 1 in which the amino acid sequencecomprises an amino acid other than S and N at the amino acid residuecorresponding to residue 96 of SEQ ID NO:2 or SEQ ID NO:4.
 10. Therecombinant polypeptide of claim 9 in which the amino acid sequence hasa G, F, Y, or I at the amino acid residue corresponding to residue 96 ofSEQ ID NO:2 or SEQ ID NO:4.
 11. The recombinant polypeptide of claim 10in which the amino acid sequence further comprises one or more of thefollowing features selected from: residue 94 is an aromatic amino acidresidue or G; residue 153 is an aliphatic amino acid residue other thanL; residue 199 is any amino acid residue other than L; residue 202 is analiphatic amino acid residue other than A; or residue 206 is an aromaticamino acid residue.
 12. The recombinant polypeptide of claim 11 in whichthe amino acid sequence comprises one or more of the following featuresselected from: residue 94 is F or G; residue 153 is G or A; residue 199is K, I, N, R, V, Q, or W; residue 202 is I, L, or G; and residue 206 isF.
 13. The recombinant polypeptide of any one of claims 9 to 12 in whichthe amino acid sequence further comprises one or more of the followingfeatures selected from: residue 49 is a polar amino acid residue otherthan K; residue 53 is an acidic amino acid residue; residue 54 is asmall or aliphatic amino acid residue other than T/P; residue 60 is analiphatic amino acid residue other than V; residue 95 is an aliphaticamino acid other than V; residue 97 is a small amino acid or G; residue109 is a basic amino acid residue other than K; residue 147 is analiphatic amino acid residue; residue 165 is a hydroxyl or small aminoacid residue; residue 197 is a small amino acid residue or G; residue223 is an aliphatic amino acid residue other than L; and residue 233 isa small amino acid residue or G.
 14. The recombinant polypeptide ofclaim 13 in which the amino acid sequence comprises on or more featuresselected from: residue 49 is R; residue 53 is D; residue 54 is A;residue 60 is A; residue 95 is L; residue 97 is G; residue 109 is R;residue 147 is L; residue 165 is T; residue 197 is G; residue 223 is V;and residue 233 is G.
 15. The recombinant polypeptide of claim 14 inwhich the amino acid sequence has one or more of the features selectedfrom: residue 54 is A; residue 109 is R; residue 147 is L; and residue233 is G.
 16. The recombinant polypeptide of claim 9 in which the aminoacid sequence comprises a G, I, C or an aromatic amino acid at the aminoacid residue corresponding to residue 96 of SEQ ID NO:2 or SEQ ID NO:4.17. The recombinant polypeptide of claim 16 in which the amino acidsequence further comprises one or more of the features selected from:residue 94 is an aromatic amino acid residue or G; residue 153 is analiphatic amino acid residue other than L; residue 199 is any amino acidresidue other than L; residue 202 is an aliphatic amino acid residueother than A; and residue 206 is an aromatic amino acid residue.
 18. Therecombinant polypeptide of claim 17 in which the amino acid sequencecomprises one or more of the features selected from: residue 94 is F orG; residue 153 is G or A; residue 199 is K, I, N, R, V, Q, or W; residue202 is I, L, or G; and residue 206 is F.
 19. The recombinant polypeptideof any one of claims 16 to 18 in which the amino acid sequence furthercomprises one or more of the features selected from: residue 49 is apolar amino acid residue other than K; residue 53 is an acidic aminoacid residue; residue 54 is a small or aliphatic amino acid residueother than T/P; residue 60 is an aliphatic amino acid residue other thanV; residue 95 is an aliphatic amino acid other than V; residue 97 is asmall amino acid or G; residue 109 is a basic amino acid residue otherthan K; residue 147 is an aliphatic amino acid residue; residue 165 is ahydroxyl or small amino acid residue; residue 197 is a small amino acidresidue or G; residue 223 is an aliphatic amino acid residue other thanL; and residue 233 is a small amino acid residue or G.
 20. Therecombinant polypeptide of claim 19 in which the amino acid sequencecomprises one or more of the features selected from: residue 49 is R;residue 53 is D; residue 54 is A; residue 60 is A; residue 95 is L;residue 97 is G; residue 109 is R; residue 147 is L; residue 165 is T;residue 197 is G; residue 223 is V; and residue 233 is G.
 21. Therecombinant polypeptide of claim 20 in which the amino acid sequence hasone or more of the features selected from: residue 54 is A; residue 109is R; residue 147 is L; and residue 233 is G.
 22. The recombinantpolypeptide of claim 1 which comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, and
 138. 23. The recombinantpolypeptide of any one of claims 1 to 21 in which the recombinantpolypeptide is an isolated polypeptide.
 24. The recombinant polypeptideof claim 23 in which the isolated polypeptide is a substantially purepolypeptide.
 25. A polynucleotide encoding the recombinant polypeptideof any one of claims 1 to
 22. 26. The polynucleotide of claim 25selected from the group consisting of SEQ ID NO: 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, and
 137. 27. Thepolynucleotide of claim 25 in which the polynucleotide hybridizes underhigh stringency condition to a polynucleotide selected from the groupconsisting of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, and
 137. 28. An expression vector comprising thepolynucleotide of any one of claims 25 and 27 operably linked to controlsequences suitable for directing expression in a host cell.
 29. Theexpression vector of claim 28 in which the control sequence is apromoter.
 30. The expression vector of claim 29 in which the promotercomprises an E. coli. promoter.
 31. The expression vector of claim 29 inwhich the control sequence is a secretion signal.
 32. A host cellcomprising the expression vector of any one of claim 28 to
 31. 33. Thehost cell of claim 32 which is homologous with the cell type of thewild-type ketoreductase enzyme from which the engineered ketoreductaseenzyme was derived.
 34. The host cell of claim 32 which is heterologouswith the cell type of the wild-type ketoreductase enzyme from which theengineered ketoreductase enzyme was derived.
 35. The host cell of claim32 in which the cell is E. coli.
 36. The host cell of claim 32 or 35 inwhich the codons comprising the expression vector have been optimizedfor expression in said host cell.